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. 2012 Aug 7;109(32):13004-9.
doi: 10.1073/pnas.1210787109. Epub 2012 Jul 23.

X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner

G Grant Welstead  1 , Menno P Creyghton, Steve Bilodeau, Albert W Cheng, Styliani Markoulaki, Richard A Young, Rudolf Jaenisch
Affiliations

X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner

G Grant Welstead et al. Proc Natl Acad Sci U S A. .

Abstract

Embryogenesis requires the timely and coordinated activation of developmental regulators. It has been suggested that the recently discovered class of histone demethylases (UTX and JMJD3) that specifically _target the repressive H3K27me3 modification play an important role in the activation of "bivalent" genes in response to specific developmental cues. To determine the requirements for UTX in pluripotency and development, we have generated Utx-null ES cells and mutant mice. The loss of UTX had a profound effect during embryogenesis. Utx-null embryos had reduced somite counts, neural tube closure defects and heart malformation that presented between E9.5 and E13.5. Unexpectedly, homozygous mutant female embryos were more severely affected than hemizygous mutant male embryos. In fact, we observed the survival of a subset of UTX-deficient males that were smaller in size and had reduced lifespan. Interestingly, these animals were fertile with normal spermatogenesis. Consistent with a midgestation lethality, UTX-null male and female ES cells gave rise to all three germ layers in teratoma assays, though sex-specific differences could be observed in the activation of developmental regulators in embryoid body assays. Lastly, ChIP-seq analysis revealed an increase in H3K27me3 in Utx-null male ES cells. In summary, our data demonstrate sex-specific requirements for this X-linked gene while suggesting a role for UTY during development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
UTX is required for development in a sex-specific manner. (A) Utx is required for development of female embryos. Representative embryos isolated at the respective time from a natural mating between a mutant male and heterozygote female (UtxKO/Wt) are shown. All images are taken at the same magnification (8×) except for the E9.5 embryos, which were taken at a higher magnification (20×). (B) A representative litter from E9.5 that demonstrates the variability in phenotypes of Utx-null female embryos. (C) UTX-deficient males are smaller than wild-type males throughout their lifespan. Error bars represent the SD of the weights taken at that age. A representative image of a Utx-null male and wild-type littermate is shown. (D) UTX-deficient males die prematurely compared with wild-type littermates. The percent survival of wild-type (n = 42) and UtxKO males (n = 22) followed for 200 d after birth.
Fig. 2.
Fig. 2.
Utx regulates the levels of H3K27me3 in ES cells and the timely activation of key developmental regulators during differentiation. (A) Representative pictures of the mesoderm, endoderm, and ectoderm structures observed in ES cell derived teratomas. UtxKO/KO ES cell-derived teratomas contained a large number of giant trophoblast cells. Cartilage (C), Respiratory Epithelium (RE), neurons (N) and ciliated structure (Arrows) are indicated. (B) H&E staining of EBs harvested at day 10 postdifferentiation. Heterozygous female ES cells display structures indicative of differentiation in essentially all EBs in view. Female UTX-null ES cells do not. (C) Gene activation during embryoid body differentiation of male UTX-deficient (UtxKO) male, UTX-deficient (UtxKO/KO) female, and UTX-heterozygote (UtxKO/Wt) female ES cells. qRT-PCR was performed on RNA harvested from ES cells and EBs collected at day 10. Fold activation over GAPDH is represented for four genes representing the three germ layers: mesoderm (Gata4 and Gata6), endoderm (T), and ectoderm (Otx2). (D) H3K27me3 increases at developmental regulators in UTX-deficient ES cells. ChIP-seq density profiles for H3K27me3 and H3K4me3 in male Utx-null (UtxKO) ES cells and male ES cells with restored UTX expression (UtxFlx) are shown. The y axis floor is set at 0.5 reads per million. Gene models are shown below the density profiles.
Fig. 3.
Fig. 3.
UTX is not required for RA-induced differentiation. (A) Increased levels of H3K27me3 at the Hoxb cluster in the absence of UTX. H3K27me3 (red) and H3K4me3 (green) gene tracks for male ES cells with restored UTX expression (UtxFlx) and Utx-null (UtxKO) male ES cells that were untreated or treated with RA. During differentiation, bivalent genes can be maintained (yellow shading) or resolved (blue shading). Maintained bivalent genes are defined as those that maintain significant levels of H3K4me3 and H3K27me3 during RA-induced differentiation. Resolved bivalent genes are defined as genes that lost H3K27me3, but retained H3K4me3. The y axis floor is set at 0.5 reads per million. Gene models are shown below the density profiles. (B) Bivalent genes in UTX-deficient ES cells have increased levels of H3K27me3 and a reduction in H3K4me3. Metagene representations of the average H3K4me3 (green lines) and H3K27me3 (red lines) levels around transcription start sites (±2 kb) for wild-type (solid lines) and Utx-deficient (dashed lines) ES cells. (C) Demethylation of H3K27me3 in UTX-deficient ES cells in response to RA. Metagene representations of the average H3K4me3 (green lines) and H3K27me3 (red lines) levels around transcription start sites (±2 kb) for wild-type (solid lines) and Utx-deficient (dashed lines) ES cells. H3K27me3 levels are reduced at resolved genes for both wild-type and UTX-deficient ES cells. (D) UTX-deficient male ES cells are transcriptionally responsive to RA. Violin plots summarizing the gene expression of wild-type (UtxFlx) and UTX-deficient (UtxKO) ES cells grown in self-renewal conditions (no RA) or under differentiation conditions (+RA). Maintained and resolved bivalent genes were analyzed.
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