Rescuing the aberrant sex development of H3K9 demethylase Jmjd1a-deficient mice by modulating H3K9 methylation balance

Abstract

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance.

Fig 1. Expression of GLP/G9a H3K9 methyltransferase complex in XY embryonic gonads at E11.5.

(A, B) Quantitative comparison of the immunofluorescence intensities of H3K9me2 (A) and H3K9me3 (B). Representative staining profiles are shown in S1 Fig. The intensities of H3K9 methylation of Jmjd1a^Δ/+ mesonephric cells were defined as 1. Data are presented as mean ± SD. *** P < 0.001; n.s., not significant. (C) Relative mRNA expression profiles of Jmjd1a, GLP, and G9a in gonadal somatic cell populations. Gonadal somatic cells were prepared from dissociated gonads from E11.5 Nr5a1-hCD271-tg embryos and fractionated according to the expression levels of hCD271 by FACS (S2 Fig). Each fraction was subjected to mRNA expression analysis by RT-qPCR. The endogenous Nr5a1 expression level was strictly correlated with the expression levels of hCD271 (left). Sry and Jmjd1a transcripts were substantially enriched in the hCD271-high population whereas GLP/G9a transcripts were detected in each population (right). mRNA expression levels in the hCD271-low population were defined as 1. Data are presented as mean ± SD. * P < 0.05, *** P < 0.001; n.s., not significant. (D, E) Triple immunofluorescence analyses for GLP (D) and G9a (E), counterstained with anti-Jmjd1a and anti-Sry in the center regions of E11.5 gonads. Enlarged boxes indicate co-expression of GLP (D) and G9a (E) with Jmjd1a in Sry-expressing pre-Sertoli cells (arrowheads). Asterisks represent germ cells. The population of the cells containing both signals of GLP (or G9a) and Jmjd1a among the Sry-expressing pre-Sertoli cells is presented at right. More than 200 cells per embryo (n = 3) were examined. Data are presented as mean ± SD. Scale bar, 50 μm.

Fig 2. GLP/G9a complex-mediated H3K9 methylation counteracts Jmjd1a-mediated H3K9 demethylation in gonadal somatic cells.

(A) Flow cytometric analyses of E11.5 gonadal somatic cells for quantifying the H3K9 methylation levels. Cells were prepared from gonads with mesonephroi and then co-stained with antibodies against H3K9me2 and Nr5a1. Upper and lower boxes indicate the populations of Nr5a1-positive gonadal somatic cells and Nr5a1-negative mesonephric cells, respectively. (B) Representative histogram analyses for H3K9me2 levels of Nr5a1-positive gonadal somatic cells (upper) and Nr5a1-negative mesonephric cells (lower). (C) Statistical analysis of H3K9me2 levels of gonadal somatic cells of the indicated genotypes. Median fluorescence intensity (MFI) values for H3K9me2 of Nr5a1-positive gonadal somatic cells were normalized to those of Nr5a1-negative cells, and then plotted (n = 4–5). * P < 0.05; ** P < 0.01.

Fig 3. GLP/G9a complex catalyzes H3K9 dimethylation at the Sry locus.

(A) Diagram of the Sry locus and primer location of the linear promoter region of Sry. (B) ChIP-qPCR analysis for G9a at the linear promoter region of Sry. Gonadal somatic cells were purified from E11.5 XY Nr5a1-hCD271-tg embryos, pooled, cross-linked, and then introduced into ChIP-qPCR analysis. We used Npas4, that had been identified as one of the target loci of G9a, as a positive control locus [16]. Data are presented as mean ± SD. * P < 0.05; *** P < 0.001. (C) ChIP-qPCR analyses for H3K9me2 (left) and H3K4me2 (right) at the Sry locus. Gonadal somatic cells of the indicated genotypes were purified according to the method described in S3 Fig, pooled for each genotype (2 to 4 embryos) and then subjected to native ChIP analysis. ChIP experiment was performed independently twice and gave similar results. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; n.s., not significant.

Fig 4. Jmjd1a- and GLP/G9a complex-mediated expression tuning is not extended to other genes on the Y chromosome.

(A) E11.5 XY gonads of the indicated genotypes were dissected and subjected to mRNA expression analysis. We examined the expression levels of Sry-neighboring genes (Uty, Ddx3y, Usp9y, and Zfy2), which are located on the short arm of the Y chromosome. There was no significant difference in the expression levels of these genes between control and mutant gonads. mRNA expression levels in the adult testis (3 months) were defined as 1. All data are presented as mean ± SD. (B) ChIP-qPCR analyses for H3K9me2 at the Uty, Ddx3y, Usp9y, and Zfy2 loci. Primer locations are shown at the top. There was no significant difference of the H3K9me2 levels between control and mutant gonads. All data are presented as mean ± SD.

Fig 5. The GLP mutation rescues the reduced expression of Sry in Jmjd1a-deficient embryos.

(A, C) Co-immunostaining profiles of Sry (A) and Sox9 (C) with a gonadal somatic cell marker, Gata4, in the center regions of E11.5 gonads of the indicated genotypes. Scale bar, 20 μm. (B, D) The ratios of cells positive for Sry (B) and Sox9 (D) to Gata4-positive cells in E11.5 gonads of the indicated genotypes. Numbers of examined animals are shown above the bars. All data are presented as mean ± SD. * P < 0.05; ** P < 0.01.

Fig 6. The GLP mutation rescues abnormal sex differentiation of XY Jmjd1a-deficient embryos.

(A) Evaluation of sex differentiation of E13.5 embryonic gonads by immunofluorescence analysis. Sox9 and Foxl2 mark testicular Sertoli and ovarian somatic cells, respectively. The enlarged box indicates that the GLP mutation rescued the tubule-like structure that was absent in XY Jmjd1a^Δ/Δ gonads. Scale bar, 200 μm. (B) Quantification of Sox9- and Foxl2-positive cells in E13.5 gonads of the indicated genitypes. Numbers of embryos examined are shown above the bars. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.

Fig 7. The GLP mutation rescues XY sex reversal in Jmjd1a-deficient adult mice.

(A) External genitalia (upper) and gonads and genital tracts (lower) of 3-months-old mice of the indicated genotypes. Arrowheads represent mammary glands. The distance between anus and penis or vagina is indicated. Frequencies are presented in the lower left corner. Te, testis; Ov, ovary. (B) Frequency analysis of abnormal sex differentiation of 3-months-old mice, determined by examining internal genitalia. Numbers of animals examined are shown above the bars.

Fig 8. Embryonic administration of the GLP/G9a inhibitor UNC0642 rescues aberrant sex development of Jmjd1a-deficient mice.

(A) Experimental scheme of UNC0642 treatment. 0.5 mg of UNC0642 was intraperitoneally injected into pregnant females carrying E10.5 Jmjd1a-deficient embryos, and the subsequent gonadal differentiation of E13.5 embryos (B) and 3-months-old adults (D) was examined. (B) Immunofluorescence analysis of sex differentiation of E13.5 embryonic gonads using antibodies against Sox9 and Foxl2. Scale bar, 200 μm. (C) Quantification of Sox9- and Foxl2-positive cells in E13.5 gonads. Numbers of embryos examined are shown above the bars. Data are presented as mean ± SD. * P < 0.05. (D) External genitalia (upper) and gonads and genital tracts (lower) of UNC0642-treated (right) and solvent-treated (left) XY Jmjd1a-deficient animals. Arrowheads represent mammary glands. The distance between anus and penis or vagina is indicated. Frequencies are presented in the lower left corner. Te, testis; Ov, ovary. (E) Frequency analysis of abnormal sex differentiation of 3-months-old mice, determined by examining the internal genitalia. Numbers of animals examined are shown above the bars.

Fig 9. Fine-tuning of Sry expression is achieved by the balance in activities between H3K9 demethylase Jmjd1a and H3K9 methyltransferase GLP/G9a complex.

In wild-type embryonic gonads, Jmjd1a removes H3K9 methylation marks, which were deposited by GLP/G9a complex, from the Sry locus, thereby ensuring Sry activation. In Jmjd1a-deficient embryonic gonads, the absence of Jmjd1a results in the GLP/G9a complex-mediated H3K9 hypermethylation at the Sry locus, thereby compromising Sry expression and causing male-to-female sex reversal. Normalization of the H3K9 methylation balance of the Sry locus by a genetic or a pharmacological approach rescues the aberrant sex development of Jmjd1a-deficient mice by restoring Sry expression.