Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development

Lambrot R, Chan D, Shao X, Aarabi M, Kwan T, Bourque G, Moskovtsev S, Librach C, Trasler J, Dumeaux V, Kimmins S., 20.07.2021

Abstract

The paternal environment has been linked to infertility and negative outcomes. Such effects may be transmitted via sperm through histone modifications. To date, in-depth profiling of the sperm chromatin in men has been limited. Here, we use deep sequencing to characterize the sperm profiles of histone H3 lysine 4 tri-methylation (H3K4me3) and DNA methylation in a representative reference population of 37 men. Our analysis reveals that H3K4me3 is localized throughout the genome and at genes for fertility and development. Remarkably, enrichment is also found at regions that escape epigenetic reprogramming in primordial germ cells, embryonic enhancers, and short-interspersed nuclear elements (SINEs). There is significant overlap in H3K4me3 and DNA methylation throughout the genome, suggesting a potential interplay between these marks previously reported to be mutually exclusive in sperm. Comparisons made between H3K4me3 marked regions in sperm and the embryonic transcriptome suggest an influence of paternal chromatin on embryonic gene expression.

Lambrot R, Chan D, Shao X, Aarabi M, Kwan T, Bourque G, Moskovtsev S, Librach C, Trasler J, Dumeaux V, Kimmins S. Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development. Cell Rep. 2021 Jul 20;36(3):109418. doi: 10.1016/j.celrep.2021.109418. PMID: 34289352.

Publication: https://doi.org/10.1016/j.celrep.2021.109418

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The publication Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development by Lambrot R, Chan D, Shao X, Aarabi M, Kwan T, Bourque G, Moskovtsev S, Librach C, Trasler J, Dumeaux V, Kimmins S. is published under an open access license: http://creativecommons.org/licenses/by-nc-nd/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.

Data set 1: H3K4me3 is enriched at CpG-rich regions, promoters, and SINEs in human sperm

Other: Chromatin Immunopreciptation Sequencing

Species

Species
Human

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001363: testis	Adult	A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity.	Human	30

Cell Types

Cell ontology	Description	Species
CL_0000018: spermatid	A male germ cell that develops from the haploid secondary spermatocytes. Without further division, spermatids undergo structural changes and give rise to spermatozoa.	Human
CL_0000017: spermatocyte	A male germ cell that develops from spermatogonia. The euploid primary spermatocytes undergo meiosis and give rise to the haploid secondary spermatocytes which in turn give rise to spermatids.	Human
CL_0000020: spermatogonium	An euploid male germ cell of an early stage of spermatogenesis.	Human
CL_0000016: male germ line stem cell	A stem cell that is the precursor of male gametes.	Human

Images

Figure 1: H3K4me3 is enriched at CpG-rich regions, promoters, and SINEs in human sperm

ChIP-seq for H3K4me3 was performed on a sample prepared from the sperm of 30 men, and peaks were called using MACS2. (A) Location of the H3K4me3 peaks based on genomic and CpG annotations and overlap with repetitive elements and low-complexity DNA sequences. Annotations were obtained from the Bioconductor package annotatr (Cavalcante and Sartor, 2017) and RepeatMasker (http://www.repeatmasker.org/). The overlap of peaks between different annotations is illustrated by connecting nodes, and the number of overlapping peaks is displayed on the bar graph shown above them. The top-10 overlaps are indicated. (B) Enrichment in H3K4me3 peaks for each specific annotation. Positive and negative enrichments are indicated by Z scores were determined with the Bioconductor package regioneR (Gel et al., 2016). For all annotations displayed, p < 0.001 by permutation tests. (C) Enrichment in H3K4me3 peaks for each repeat annotation; p < 0.001 by permutation tests. (D and E) Boxplots showing the level of expression of genes with promoters bearing no H3K4me3 in sperm, marked by H3K4me3 at promoters located in CpG-poor or -rich regions, and promoters overlapping with SINEs, low-complexity repeats, or hESCs enhancers during spermatogenesis (D) and development (E). The level of transcription was visualized based on the transcript abundance expressed as transcripts per million (TPM) for spermatogenesis in spermatogonial stem cells (SSCs), spermatogonia (mitotic phase), spermatocytes (meiosis), and spermatids (spermiogenesis) or reads per kilobase of exon per million (RPKM) fragments mapped for development. The p values were determined by pairwise Wilcoxon rank-sum tests with a Benjamini-Hochberg correction for multiple testing. NS indicates a non-significant adjusted p value. ∗adjusted p < 0.05, ∗∗∗adjusted p < 0.001. See also Figures S1–S4 and Table S1.

Source: Lambrot R, Chan D, Shao X, Aarabi M, Kwan T, Bourque G, Moskovtsev S, Librach C, Trasler J, Dumeaux V, Kimmins S. Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development. Cell Rep. 2021 Jul 20;36(3):109418. doi: 10.1016/j.celrep.2021.109418. PMID: 34289352.

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/

Figure 2: Overlap between sperm H3K4me3 peaks and CpG-rich regions, repetitive elements, and hESC enhancers

(A–F) Genome-browser snapshots showing the reference-population ChIP-seq track (blue), H3K4me3 peaks called by MACS2 (dark blue boxes), a CpG island location track (red boxes, UCSC), a GC percentage heatmap (blue, low GC percentage; white, 50% GC; red, high GC percentage), the location of SINEs (dark gray boxes), low-complexity repeats (dark purple boxes), the location of LINEs (violet boxes) (obtained from RepeatMasker), and the location of hESC enhancers (light blue boxes) (Barakat et al., 2018). The overlap of H3K4me3 with each of these elements is illustrated for the member of the Argonaute family of genes implicated in RNA-mediated gene silencing AGO4 (A), for two genes coding for tubulin isoforms TUBA4A and TUBA4B (B), for the gene assisting in double-strand breaks repair RAD51 (C), for the developmental genes ID4 (D) and SOX11 (E), and, finally, for a random intergenic region (F).

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 2: H3K4me3 and DNA methylation co-occur at functional genomic regions in human sperm

Methylome: Whole Genome Bisulfite Sequencing

Species

Species
Human

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001363: testis	Adult	A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity.	Human	30

Cell Types

Cell ontology	Description	Species
CL_0000016: male germ line stem cell	A stem cell that is the precursor of male gametes.	Human
CL_0000020: spermatogonium	An euploid male germ cell of an early stage of spermatogenesis.	Human
CL_0000017: spermatocyte	A male germ cell that develops from spermatogonia. The euploid primary spermatocytes undergo meiosis and give rise to the haploid secondary spermatocytes which in turn give rise to spermatids.	Human
CL_0000018: spermatid	A male germ cell that develops from the haploid secondary spermatocytes. Without further division, spermatids undergo structural changes and give rise to spermatozoa.	Human

Images

Figure 3: DNA methylation levels in regions with H3K4me3 peaks in human sperm

WGBS was performed on the sperm of the same 30 men used for H3K4me3 ChIP-seq, and the DNA methylation levels of the CpGs located within H3K4me3 peaks were assessed. The average DNA methylation level of the regions marked by peaks was then calculated. (A) Number of H3K4me3 peaks with low (hypomethylated, ≤20% methylation), intermediate (20% ≤ methylation ≤ 80%), or high DNA methylation (hypermethylated, ≥80% methylation). (B) Heatmaps showing the H3K4me3 signal (in RPKM) and the corresponding DNA methylation levels (in %) around the center of the peaks (±5 kb) for the hypomethylated (23,023 peaks), intermediate (8,087), or hypermethylated (16,777) H3K4me3 peaks. Each line represents a H3K4me3 peak. The overall H3K4me3 signal and DNA methylation for all peaks are summarized on top of the heatmaps. (C) Proportion of hypomethylated, intermediate, and hypermethylated DNA within H3K4me3 peaks for genomic, CpG, RNAs, and repeats annotations.

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/

Figure 4: H3K4me3 and DNA methylation profiles in human sperm

(A–C) The overlap between H3K4me3 peaks and DNA methylation is displayed in genome-browser snapshots showing the ChIP-seq track (blue), H3K4me3 peaks called by MACS2 (dark blue), the WGBS track (black), a CpG islands location track (red, UCSC), and a GC percentage heatmap (blue, low GC %; white, 50% GC; red, high GC %) for the HOXA developmental gene cluster (A), for the chromatin modifier ARID5B (B), for the spermatogonial marker ZBTB16/PLZF (C), and for the spermiogenesis gene CYLC2 (C). H3K4me3 peaks located at non-DNA methylated CpG islands (yellow), at regions including various degrees of methylation (purple), at CpG-poor DNA methylated regions (green), and at regions of intermediate DNA methylation (gray) are highlighted.

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 3: The functional relationship of sperm H3K4me3 and DNA methylation in gene regulation during spermatogenesis and embryogenesis

Methylome: Whole Genome Bisulfite Sequencing

Species

Species
Human

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001363: testis	Adult	A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity.	Human	30

Cell Types

Cell ontology	Description	Species
CL_0000016: male germ line stem cell	A stem cell that is the precursor of male gametes.	Human
CL_0000020: spermatogonium	An euploid male germ cell of an early stage of spermatogenesis.	Human
CL_0000017: spermatocyte	A male germ cell that develops from spermatogonia. The euploid primary spermatocytes undergo meiosis and give rise to the haploid secondary spermatocytes which in turn give rise to spermatids.	Human
CL_0000018: spermatid	A male germ cell that develops from the haploid secondary spermatocytes. Without further division, spermatids undergo structural changes and give rise to spermatozoa.	Human

Images

Figure 5: DNA methylation levels vary within H3K4me3 peaks at gene promoters with functions in spermatogenesis, embryogenesis, and basic cellular processes

(A) Over-representation of H3K4me3 peaks with hypo-, intermediate-, and hyper- average DNA methylation in the promoter of genes implicated in spermatogenesis. The genes relevant to early spermatogenesis, meiosis, and spermiogenesis were selected from two databases: Db1 (Jan et al., 2017) and Db2 (Wang et al., 2018). Benjamini-Hochberg-adjusted hypergeometric minimum-likelihood p values are indicated. (B) Gene ontology analysis of the H3K4me3 peaks located in gene promoters (1 kb upstream of the TSS). The enrichment in a specific biological process was measured by PANTHER over-representation test (GO biological process complete). The p value was determined by Fisher’s exact test with a Bonferroni correction for multiple testing. The top-10 processes with the lowest p value are indicated after filtering for non-redundant processes with an enrichment ≥1.25. Full list of GO-enriched terms is provided in Table S4.

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/

Figure 6: H3K4me3/H3K27me3 bivalency at H3K4me3 sperm peak regions with a hypo-, intermediate, or hyper- DNA methylation

Heatmaps showing the H3K4me3 and H3K27me3 signal (in RPKM) around the center of the H3K4me3 peaks (±5 kb) in human sperm (reference population of 30 men for H3K4me3 and published data for H3K27me3 (Hammoud et al., 2009)), eight-cell human embryo and inner cell mass (Xia et al., 2019), hESC (Grandy et al., 2015), and human fetal organs (Yan et al., 2016). Each lane represents an H3K4me3 peak region identified in the reference population with a DNA hypo- (23,023), intermediate- (8,087), hyper- (16,777), or unknown (with no CpG or CpGs that had less than 10× coverage in WGBS) methylation, or a H3K27me3 sperm peak region not marked by H3K4me3. See also Figure S7.

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 4: H3K4me3 in sperm localizes to enhancers and regions escaping epigenetic reprogramming

Other: Chromatin Immunopreciptation Sequencing

Species

Species
Human

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001363: testis	Adult	A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity.	Human	30

Cell Types

Cell ontology	Description	Species
CL_0000016: male germ line stem cell	A stem cell that is the precursor of male gametes.	Human
CL_0000020: spermatogonium	An euploid male germ cell of an early stage of spermatogenesis.	Human
CL_0000017: spermatocyte	A male germ cell that develops from spermatogonia. The euploid primary spermatocytes undergo meiosis and give rise to the haploid secondary spermatocytes which in turn give rise to spermatids.	Human
CL_0000018: spermatid	A male germ cell that develops from the haploid secondary spermatocytes. Without further division, spermatids undergo structural changes and give rise to spermatozoa.	Human

Images

Figure 7: H3K4me3 is enriched at hESC enhancers and at regions that escape reprogramming

(A) Enrichment in H3K4me3 peaks at identified hESC enhancers and super-enhancers (Barakat et al., 2018), at metastable epialleles (Kessler et al., 2018), and in regions escaping reprogramming through loss of DNA methylation in human primordial germ cells (PGCs) (Tang et al., 2015). Positive enrichments are indicated by Z scores determined by the Bioconductor package regioneR. For all regions displayed, p < 0.001 by permutation tests. (B) Proportion of hypomethylated, intermediate, and hypermethylated H3K4me3 peaks intersecting or not with the regions of interest. Changes in the proportion of peaks not intersecting the regions of interest and peaks intersecting were tested by χ2 test, and an asterisk indicates p < 0.001.

Licensed under: http://creativecommons.org/licenses/by-nc-nd/4.0/