Tet1 controls meiosis by regulating meiotic gene expression

Abstract

Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps, including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA. Although several epigenetic regulators, such as Dnmt3l and the histone methyltransferases G9a and Prdm9, have been reported to be crucial for meiosis, little is known about how the expression of meiotic genes is regulated and how their expression contributes to normal meiosis. Using a loss-of-function approach in mice, here we show that the 5mC-specific dioxygenase Tet1 has an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation that takes place in primordial germ cells, but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus establishes a function for Tet1 in meiosis and meiotic gene activation in female germ cells.

Figure 1. Subfertility of Tet1^Gt/Gt mice is associated with oocyte loss in late embryonic stage

(a) Abnormal ovaries of the Tet1^Gt/Gt mice. Top panel, representative images of ovaries from 8-week-old wild-type (Wt) and Tet1 mutant (Gt/Gt) mice. Both left and right ovaries from one representative female are shown. Bottom panel, representative images of Hematoxylin and Eosin staining of adult ovary sections. Arrows indicate fully-grown oocytes. (b) Number of fully-grown oocytes in adult ovaries. n=4 or 5. Error bars indicate SEM. **P<0.01. (c) The average number of ovulated oocytes per female after hormonal stimulation. n=5–7. Error bars indicate SEM. *P<0.05. (d) The relative oocyte numbers normalized to that in the wild-type (Wt) mouse. The number of oocytes in Wt mice was counted and set as 1. n=2–7. Error bars indicate SEM. *P<0.05, **P<0.01.

Figure 2. Meiotic defects in Tet1^Gt/Gt oocytes

(a) Distribution of E16.5, E17.5 and E18.5 oocytes in the four substages of meiotic prophase. Error bars indicate SEM. n=2–5. *P<0.05, **P<0.01, compared between +/Gt and Gt/Gt. (b) Left, representative images of zygotene oocytes co-stained with SYCP3, SYCP1, and CREST antibodies. Right, percentage of the AE-alignment impaired oocytes at the zygotene stage. Oocytes that contain less than five SYCP1-foci are counted as AE-alignment impaired. n=6–8. Error bars indicate SEM. **P<0.01. (c) Left, representative images of pachytene stage oocytes co-stained with SYCP3, SYCP1, and CREST antibodies. Right, distribution of oocyte types categorized by the numbers of univalent chromosomes in each pachytene stage oocyte. n=6–7. Error bars indicate SEM. *P<0.05. **P<0.01. (d) Representative images of oocytes co-stained with antibodies against γH2AX, SYCP3, and CREST (pachytene stage), or γH2AX, SYCP3, and MLH1 (early diplotene stage). (e) Distribution of oocyte types categorized by the staining pattern of γH2AX in each pachytene and early diplotene stage oocyte. Representative images for each group are shown in supplementary figure 9. n=3–7. Error bars indicate SEM. **P<0.01, compared to control.

Figure 3. Tet1 activates meiotic genes through DNA demethylation

(a) Scatter plot comparing transcriptome of wild-type and Tet1^Gt/Gt E13.5 female PGCs. There are 111 and 899 genes that are respectively up- or down-regulated (FDR<0.05). Examples of down-regulated meiotic genes include Mael, Sycp3, Stra8, Sycp1, and Prdm9 indicated. (b) Gene ontology analysis of down-regulated genes in Tet1^Gt/Gt PGCs with cutoff FDR<0.05. The most enriched biological processes based on their p-values are shown. (c) ChIP-qPCR analysis of E13.5 wild-type female PGCs using anti-Tet1 antibody demonstrates binding of Tet1 to the promoters of Sycp1, Mael, and Sycp3. Upper panels, diagrams of Sycp1, Mael, and Sycp3 genes with the analyzed regions indicated by red lines. Bottom panels, relative enrichment of Tet1 over IgG control. n=3. Error bars indicate SEM. *P<0.05. **P<0.01.

Figure 4. Whole genome bisulfite analysis of the effect of Tet1 knockout on DNA methylation in PGCs

(a) Wt E13.5 female PGCs are globally hypomethylated and depletion of Tet1 only slightly increased the global DNA methylation level. Shown are the DNA methylation levels in the entire mouse genome as well as the various genomic regions. In the last column, the DNA methylation levels at the Tet1 bound regions identified in mES cells were compared. (b) Heat map of the 255 differentially expressed and DMR associated genes. (c) Bisulfite sequencing analysis of the Sycp1, Mael, and Sycp3 gene promoters of Tet1 binding site in wild-type and Tet1^Gt/Gt PGCs. Each CpG is represented by a circle. Open and filled circles represent unmethylated or methylated, respectively. Percentages of DNA methylation are indicated.