Destabilization of mRNAs enhances competence to initiate meiosis in mouse spermatogenic cells

ABSTRACT The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis.

Total number of expressed genes (nFeature) A Figure S2: Features and counts in scRNA-seq data.
A: Total number of genes expressed (nFeature) and transcripts detected (nCount) in wild-type and Meioc-null cells for germ cell clusters.B: Total number of genes expressed (nFeature) and transcripts detected (nCount) in wild-type and Meioc-null cells for somatic clusters.A: Wild-type expression levels of Mki67, Meiosin, Rec8, and Stra8 for all germ cell clusters.Black and red asterisks designate enrichment or depletion, respectively, relative to all other germ cells.Clusters marked as "not done" (n.d.) did not meet expression thresholds set for statistical testing.
B: Heatmap of the top 10 enriched genes in the G1, early S, and late S preleptotene clusters.
C: Gene Set Enrichment Analysis (GSEA) for Gene Ontology (GO) terms for genes within each celltype cluster.Genes were ranked by the signed -log10(P value) for their level of enrichment/depletion within each cluster relative to all other germ cell clusters.Top three enriched GO terms for each cluster shown.A: Developmental synchronization of spermatogenesis for bulk RNA-seq analysis.IHC, immunohistochemistry; RA, retinoic acid.B: Enrichment for preleptotene spermatocytes in developmentally synchronized testes.
C: Differential expression of bulk RNA-seq data.Blue marks genes that are upregulated in response to MEIOC (log 2 fold change>0 and adjusted P<0.05).Orange marks genes that are downregulated in response to MEIOC (log 2 fold change<0 and adjusted P<0.05).Rbm46 and Stra8 are highlighted in dark gray; Meioc, Ythdc2, and Meiosin expression are highlighted in dark blue.Other dark blue genes represent those that fall under meiosis-associated GO terms from panel E. D: Cell cycle analysis of differential expression from bulk RNA-seq data.MEIOC increases abundance of genes associated with G1/S, S, and G2 phases, similar to STRA8.WT           A: Normalized counts in WT vs. Meioc KO bulk RNA-seq analysis of preleptotene-enriched testes for E2f6, Mga, and other subunits of PRC1.6.B: Percentage of E2F6-repressed and -activated, as well as MGA-repressed and -activated, genes among MEIOC-upregulated, MEIOC-downregulated, and expressed genes from bulk RNA-seq analysis of preleptotene-enriched testes.E2F6 and MGA targets as defined in Figure S8C.
B: Left panel, correlation between MEIOC-dependent changes from bulk RNA-seq analysis of preleptotene-enriched testes and STRA8-dependent changes from bulk RNA-seq analysis of sorted preleptotene spermatocytes.Analysis was limited to genes that were statistically dependent on STRA8 (adjusted P<0.05).Right panel, distribution of correlations for gene sets randomly sampled from genes expressed in the two bulk RNA-seq datasets.
C: Percentage of STRA8-activated genes in MEIOC-upregulated, and expressed genes from bulk RNA-seq analysis of preleptotene-enriched testes.STRA8-activated genes were identified as those genes with STRA8-bound promoters (as identified by Kojima et al. (2019)
Figure S10 Figure S11: MEIOC does not affect DMRT1 expression or DMRT1-dependent signaling during the mitosis-to-meiosis transition.A: Expression levels of Dmrt1 in wildtype vs. Meioc-null cells in all germ cell clusters identified in the scRNA-seq analysis.Cluster marked as "not done" (n.d.) did not meet expression thresholds set for statistical testing.B: Normalized counts of Dmrt1 in bulk RNA-seq anlaysis of wildtype vs. Meioc-null preleptotene-enriched testes.
Figure S8: MEIOC-YTHDC2-RBM46's repression of E2f6 and Mga mRNA relieves E2F6-and MGA-mediated transcriptional repression.A: Input-subtracted E2F6 ChIP-seq signal from wild-type and E2F6 KO ESCs as well as normalized MGA and IgG ChIP-seq signal from wild-type ESCs at the promoters of Meiosin, Stra8, Meioc, Rbm46, and Ythdc2.Called peaks are marked by a red bar above.Transcriptional start sites are marked by red arrows.E2F6 ChIP-seq data were reanalyzed from Dahlet et al., 2021; MGA ChIP-seq data were reanalyzed from Stielow et al., 2018.E2f6-dependent and Mga-dependent differential expression program in mouse embryonic stem cells.RNA-seq data were reanalyzed from Dahlet et al., 2021 and Qin et al., 2021.Log 2 fold change was defined as WT/KO.Genes upregulated or downregulated by E2F6 or MGA are shown in blue and orange, respectively.Gray represents genes that do not change expression in response to E2F6 or MGA.C: Overlap of E2F6-repressed and -activated genes with MGA-repressed and -activated genes, identified from reanalysis of published ChIP-seq and RNA-seq datasets from mouse embryonic stem cells.D: Percentage of E2F6-and/or MGA-regulated genes among MEIOC-upregulated, MEIOC-downregulated, and expressed genes, as identified via scRNA-seq analysis.Each E2F6/MGA gene set was tested for enrichment among MEIOC-upregulated genes and MEIOC-downregulated genes relative to expressed genes.E2F6-regulated genes and MGA-regulated genes were identified as shown in panel C.
Meiosin gene expression in response to retinoic acid.A: MEIOSIN protein expression in STRA8-positive preleptotene spermatocytes from wild-type and Meioc KO P10 testes.Arrowheads represent STRA8-positive spermatocytes with particularly robust MEIOSIN that are absent from Meioc KO testes.Graph represents MEIOSIN median intensity from three WT and Meioc KO littermate pairs.75 cells were quantified per testis.Scale bar = 20 um.B: Expression levels of RAR and RXR genes in wildtype vs. Meioc-null cells in all germ cell clusters identified in the scRNA-seq analysis.Rarb and Rxrg were not detected as expressed.Color legend same as in panel C. Clusters marked as "not done" (n.d.) did not meet expression thresholds set for statistical testing.C: Normalized counts of RAR and RXR genes as well as Rec8 in bulk RNA-seq analysis of wildtype vs. Meioc-null preleptotene-enriched testes.Rarb and Rxrg were not detected as expressed.D: Expression levels of Rec8 in wildtype vs. Meioc-null cells in all germ cell clusters identified in the scRNA-seq analysis.Color legend same as in panel C. Clusters marked as "not done" (n.d.) did not meet expression thresholds set for statistical testing., adj.P<0.05; **, adj.P<0.01; ***, adj.P<0.001; ****, adj.P<0.0001; n.s., not significant; n.t., not tested (comparison was excluded from statistical testing because log 2 fold change> -0.1 and <0.1); n.d., not detected (transcript expressed in <25% cells in each population being compared).
Crabp2 was not detected as expressed.
via STRA8-FLAG ChIP-seq in preleptotene-enriched testes) and upregulated by STRA8 (as identified by reanalysis of bulk RNA-seq data from wild-type and Stra8 KO sorted preleptotene spermatocytes fromKojima et al., 2019).D. Motif enrichment within promoters of MEIOC-upregulated genes from bulk RNA-seq analysis of preleptotene-enriched testes.E: Percentage of genes repressed by both E2F6 and MGA; E2F6 only; and MGA only in STRA8-activated and all expressed genes.Genes repressed by E2F6 and MGA were defined in mouse embyronic stem cells as shown in FigureS8C.F: Percentage of genes that are activated by STRA8 and repressed by E2F6 or MGA; and activated by STRA8 but not repressed by E2F6 or MGA among MEIOC-upregulated and expressed genes from bulk RNA-seq analysis of preleptotene-enriched testes.Genes repressed by E2F6 or MGA are defined as those that are repressed by both E2F6 and MGA; by E2F6 only; or by MGA only, as shown in FigureS8C.