Teperek M et al. (2016), Sperm is epigenetically programmed to regulate ...

XB-ART-53997

Genome Res 2016 Aug 01;268:1034-46.

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Sperm is epigenetically programmed to regulate gene transcription in embryos.

Teperek M , Simeone A , Gaggioli V , Miyamoto K , Allen GE , Erkek S , Kwon T , Marcotte EM , Zegerman P , Bradshaw CR , Peters AH , Gurdon JB , Jullien J .

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For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.

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Species referenced: Xenopus
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	Figure 1. Xenopus sperm is better at supporting development than a spermatid or a somatic cell. (A) Experimental design for the generation of cloned embryos. The somatic nucleus of a gastrula cell is transplanted to a UV-enucleated egg. The resulting embryos are scored at the gastrulation and tadpole stage. (B) Scoring of embryos as % of gastrulae and as % of swimming tadpoles to the total number of cleaved embryos. Average of n = 6 independent experiments (sperm ICSI), and n = 3 independent experiments (embryo cell NT). The total number of embryos analyzed is shown above the graph. Error bars: SEM. () P-value < 0.05 (χ2 test). (C) Experimental design for the generation of sperm- and spermatid-derived embryos. Permeabilized sperm or spermatids are injected to the cytoplasm (ICSI) of an unfertilized egg. The resulting embryos are scored at the gastrulation and tadpole stage. (D) Representative images of sperm- and spermatid-embryos. Scale bars = 1 mm. (E) Scoring of embryos as % of gastrula and as % of swimming tadpoles to the total number of cleaved embryos (average of n = 6 independent experiments). The total number of embryos analyzed is shown above the graph. Error bars: SEM. () P-value < 0.05 (χ2 test).
	Figure 2. Spermatids are as good as sperm at DNA replication. (A) Sperm and spermatids are separately incubated with egg extracts supplemented with biotin-dUTP. Subsequently, DNA fibers are isolated and subjected to molecular combing, which reveals replication on single DNA fibers. (B) Examples of DNA fibers after immunostaining procedure. Antibody staining against DNA reveals the total length of the fiber (green) and antibody staining against biotin reveals the replicated DNA (red). The bottom panels show representative examples of replication staining from sperm and from spermatids incubated in egg extracts. (C) Replication extent measured as the proportion of DNA that incorporated biotin-dUTP to the total fiber length. Results are from at least 125 independent DNA fibers (22,000 kb of DNA for each sample). Error bars: SEM. Samples were not significantly different (P-value = 0.41, KS-test).
	Figure 3. Transcription of developmentally important genes is misregulated in spermatid-derived embryos compared to sperm-derived embryos. (A) Schematic representation of paternally derived haploid embryos generated by UV enucleation of eggs followed by intra-cytoplasmic sperm injection (ICSI). (B) Developmental advantage of sperm over spermatid is maintained in haploid embryos. Embryos were scored as the % of embryos reaching a gastrula stage and a swimming tadpole stage to the total number of cleaved embryos (average of n = 3 independent experiments). Numbers of embryos analyzed are indicated above the bars. Error bars: SEM. (*) P-value < 0.05 (χ2 test). (C) Genes important for development are misregulated (mostly up-regulated) in spermatid-derived embryos. Heat map representing log fold-change in expression levels of the 100 genes (rows) misregulated in spermatid versus sperm gastrula embryos (FDR < 0.05; red: up-regulated; blue: down-regulated in spermatid) across seven independent experiments (columns). (D) Developmentally important gene ontology terms enriched in the list of misregulated genes (P-value < 0.05). (E) Up-regulation of genes in spermatid-derived embryos does not correlate with their transcription in spermatid. Density scatter plot showing gene expression in spermatid-derived embryos versus that in spermatids. No correlation is observed between the two parameters for all genes (r = 0.06) as well as for the misregulated genes (red dots, r = −0.17).
	Figure 4. Genes that are misregulated in spermatid-derived embryos have different epigenetic features in sperm and spermatid. (A) Genome-wide average nucleosome occupancy at the TSS of sperm (blue) and spermatid (green) genes. (B) Boxplots showing genome-wide DNA methylation levels at the TSS ± 1 kb of sperm (blue) and spermatid (green) genes. Inset shows correlation between the DNA methylation levels of sperm and spermatid (R = 0.8, P-value < 0.05); red line: regression; dotted line: diagonal. (C) Percentage of genes harboring H3K27me3, H3K4me3, H3K4me2, or H3K9me3 peaks genome-wide (GW) and at misregulated genes (Mis). () P-value < 0.05 (χ2 test). (D) Heat maps representing H3K27me3, H3K4me3, H3K4me2, and H3K9me3 overall levels (see Supplemental Material and Supplemental Fig. S8) at misregulated genes in sperm (first column) and spermatid (second column). Each map is sorted according to the signal in spermatid. Boxplots show the distribution of methylation levels across misregulated genes. () P-value < 0.05 (KS-test) (Supplemental Table S7).
	Figure 5. H3K27me3 target genes that lose H3K4me2/3 in sperm compared to spermatids are misregulated in spermatid-derived embryos. (A,B) Differential gene expression between sperm- and spermatid-derived embryos best correlates with differential H3K4me2/3 and H3K27me3 marking in sperm and spermatids. Partial correlation network between all tested epigenetic features of the paternal chromatin (A, sperm; B, spermatid) and gene expression in the corresponding embryos. Edges (lines) represent positive (red) or negative (blue) partial correlations. Edges thickness: strength of the partial correlations. (C) H3K4me2 and H3K27me3 marking on misregulated genes is conserved between Xenopus and human sperm. As compared to all orthologs, the misregulated orthologs are enriched for H3K27me3 marks over the genome-wide average in human sperm (χ2 test, [*] P-value < 0.05). No statistical enrichment for H3K4me2 on misregulated genes as compared to the genome-wide average is observed in human sperm.
	Figure 6. Paternal genome marking by H3K4me2/3 and H3K27me3 is required for gene expression in the embryos. (A) Histone demethylase expression assay. (B) MA plot showing log fold-change (logFC, y-axis) in gene expression between Kdm5b (H3K4me2/3 demethylase)- versus control mRNA-injected embryos, against log counts per million (logCPM, x-axis). Red dots: genes differentially expressed (FDR < 0.05); N = 4 independent experiments. (C) Venn diagram of down-regulated genes upon KDM5B expression in sperm- (blue) and spermatid-derived (green) embryos. (D) Percentages of genes down-regulated upon KDM5B expression in embryos that show H3K4me2/3 and H3K27me3 promoter peaks in the paternal cell. () P-value < 0.05 (χ2 test); ↑: over-represented when compared to genome-wide distribution. (E) Proportion of misregulated genes affected in each demethylase expression assay. () P-value < 0.05 (χ2 test). (F) Same as B for KDM6B (H3K27me3 demethylase) expression. (G) Same as C with genes up-regulated upon KDM6B expression. (H) Same as D for genes up-regulated upon KDM6B expression. (I) Model of epigenetic programming of sperm for the regulation of embryonic transcription.

References [+] :

Brykczynska, Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. 2010, Pubmed