Placental sex-dependent spermine synthesis regulates trophoblast gene expression through acetyl-coA metabolism and histone acetylation

Abstract

Placental function and dysfunction differ by sex but the mechanisms are unknown. Here we show that sex differences in polyamine metabolism are associated with escape from X chromosome inactivation of the gene encoding spermine synthase (SMS). Female placental trophoblasts demonstrate biallelic SMS expression, associated with increased SMS mRNA and enzyme activity. Polyamine depletion in primary trophoblasts reduced glycolysis and oxidative phosphorylation resulting in decreased acetyl-coA availability and global histone hypoacetylation in a sex-dependent manner. Chromatin-immunoprecipitation sequencing and RNA-sequencing identifies progesterone biosynthesis as a target of polyamine regulated gene expression, and polyamine depletion reduced progesterone release in male trophoblasts. The effects of polyamine depletion can be attributed to spermine as SMS-silencing recapitulated the effects on energy metabolism, histone acetylation, and progesterone release. In summary, spermine metabolism alters trophoblast gene expression through acetyl-coA biosynthesis and histone acetylation, and SMS escape from X inactivation explains some features of human placental sex differences.

Fig. 1. Sex differences in polyamine metabolism are associated with XCI escape of SMS.

a Experimental workflow to assess XCI escape of SMS in single nuclei in female placental tissues. Nuclei were isolated from placental tissues and FACS sorted into 96 well plates with one nucleus per well. In the same well, nuclei were lysed, DNAse treated and mRNA reverse transcribed to cDNA, including a preamplification of the regions of interest. The resulting cDNA was then used for SNP typing using multiplexed TaqMan probes specific for both the reference and alternative alleles and allelic discrimination performed in each nuclei by qPCR. b Monoallelic and biallelic expression of SMS (XCI escapee) and TIMM17B (XCI inactivated gene) SNPs in single placental nuclei. c SMS mRNA levels in PHTs. d Polyamine metabolite N1,N12-diacetylspermine concentrations in PHTs. Bar graphs show mean ± SEM, N = 9–10 male PHTs and N = 9–10 female PHTs. The panel (a) was created with Biorender.com.

Fig. 2. Correlations between TCA cycle intermediates and polyamine end metabolites.

Scatter plots of TCA cycle intermediates versus (a) DAS or (b) NAS in human placental tissues. Metabolite concentrations measured by LC-MS were log transformed and Pearson’s correlation analyses performed. r = Pearson’s correlation coefficient. P-adj = P-values corrected for FDR by Benjamini–Hochberg method. DAS: N1,N12-diacetylspermine; NAS: N1-acetylspermidine. N = 59 male placentas and N = 51 female placentas.

Fig. 3. Polyamine metabolism is linked to TCA cycle metabolism.

a Illustration of the TCA cycle indicating the metabolites and enzymes measured. b TCA cycle enzyme mRNA measured by Q-PCR and (c) TCA cycle metabolites measured by LC-MS following DFMO-mediated polyamine depletion in PHTs. Bar graphs show mean ± SEM, N = 8–10 male PHTs and N = 8–10 female PHTs.

Fig. 4. Suppression of cellular energy metabolism by polyamine depletion.

Polyamine depletion by DFMO decreases (a) OXPHOS and (b) glycolysis in male PHTs. Inhibition of spermine synthesis by SMS-silencing decreases (c) OXPHOS and (d) glycolysis in both male and female PHTs. Induction of SSAT-mediated polyamine catabolism by DENSPM decreases (e) OXPHOS and (f) glycolysis in both male and female PHTs. Bar graphs show mean ± SEM, N = 8–10 male PHTs and N = 8–10 female PHTs. OCR oxygen consumption rate; ECAR extracellular acidification rate; were measured on a Seahorse bioanalyzer.

Fig. 5. Polyamine depletion reduces acetyl-coA levels and decreases histone acetylation.

In male PHTs, DFMO decreases a acetyl-coA levels as measured by LC-MS and b histone acetylation as determined by immunoblotting. c H3K27Ac abundance is decreased by DENSPM (DEN) and reversed by SAT1 silencing. d H3K27Ac abundance is decreased following SMS-silencing. Bar graphs show mean ± SEM, N = 8–10 male PHTs and N = 8–10 female PHTs.

Fig. 6. Polyamine depletion mediates global histone hypoacetylation and progesterone biosynthesis.

a H3K27Ac enrichment near transcription start sites is decreased in male PHTs following DFMO treatment. Orange line represents vehicle and blue line represents DFMO treated PHTs. b Unsupervised principal component analysis of H3K27Ac binding to genomic regions in vehicle and DFMO treated male and female PHTs. c Overlap of DBR annotated genes in H3K27Ac ChIP-seq analysis and DEG in RNA-seq analysis. d Custom views of differentially acetylated region in vehicle and DFMO treated male PHTs. Polyamine depletion decreased e HSD3B1 mRNA and f progesterone secretion in male PHTs. ChIP-seq experiments performed in N = 6 male PHTs and N = 5 female PHTs; progesterone secretion measured in N = 5 male PHTs and N = 5 female PHTs. Male and female samples are coloured blue and red respectively.

Fig. 7. Schematic overview of polyamine metabolism and the mechanism of polyamine-mediated gene expression.

a Polyamine metabolic pathway with key enzymes highlighted and the downstream effects on trophoblast function. Spermine regulates cellular energy metabolism to increase acetyl-coA availability for histone acetylation and gene expression. Inhibition of ODC with DFMO, silencing of SMS via siRNAs, or activating SSAT using DENSPM decreases spermine levels leading to dysregulation of energy metabolism, histone acetylation, and gene expression. b Model of the effects of sex-biased polyamine metabolism on trophoblast function. Top panel: under physiological conditions, the female-biased SMS expression results in modest changes to spermine synthesis which are unlikely to result in sex-biased effects of polyamines on trophoblast function. Middle panel: Under polyamine limiting conditions the female bias in spermine synthesis buffers the effects of polyamine depletion leading to marked sex differences in spermine levels and sex-specific regulation of trophoblast function. Bottom panel: Upon spermine depletion, the effects on metabolism and gene expression become apparent but the sex-dependent effects are not observed because the source of the sex-bias is no longer present. Spermine levels are represented by the pie-charts.