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First published online 26 November 2008
doi: 10.1242/dev.026427

Development 136, 139-146 (2009)
Published by The Company of Biologists 2009
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A proximal conserved repeat in the Xist gene is essential as a genomic element for X-inactivation in mouse

Yuko Hoki1,*, Naomi Kimura1,*, Minako Kanbayashi1, Yuko Amakawa1,2, Tatsuya Ohhata1, Hiroyuki Sasaki1,2 and Takashi Sado1,2,{dagger}

1 Division of Human Genetics, National Institute of Genetics, Research Organization of Information and Systems, 1111 Yata, Mishima 411-8540, Japan.
2 Department of Genetics, The Graduate University for Advanced Studies (Sokendai), 1111 Yata, Mishima 411-8540, Japan.


Figure 1
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  		Fig. 1. Targeted deletion of the A-repeat in the Xist gene. (A) Scheme for generating the Xist{Delta}A allele. Genomic structure of the Xist gene and the targeting vector are shown. The A-repeat region is shown in gray. The Xist{Delta}A allele lacks an 812 bp fragment containing the A-repeat (nucleotides 101-912 in GenBank L04961). Positions of the probes used for Southern blotting in B and C are also indicated. B, BamHI; P, PstI; R, EcoRI; Xb, XbaI; Xh, XhoI. (B) Homologous recombination was confirmed by Southern blotting. {Delta}A2lox211 is one of the four ES lines harboring the correct targeting event. Genomic DNA digested with XbaI (left) and PstI (right) was probed with HS0.7 (Sado et al., 2005Go) and XXh0.7 (Sado et al., 2006Go), respectively. (C) The presence of the respective mutation in the mouse was confirmed by Southern blotting. Tail DNA digested with XbaI was probed with BE0.6 (Sado et al., 2005Go).

 

Figure 2
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  		Fig. 2. Expression of Xist and Tsix in Xist{Delta}A/Y ES cells. (A) The stable level of Xist and Tsix RNA was quantitated by real-time RT-PCR. Xist expression in Xist{Delta}A/Y ES cells was comparable to that in the parental wild-type male ES cells before and after differentiation (upper panel). There was a striking difference in the expression level of Tsix in Xist{Delta}A/Y and wild-type XY ES in the undifferentiated state (lower panel). The former was about 5-fold higher than the latter, although both were downregulated upon differentiation. (B) The stability of Xist and Tsix RNA was analyzed by treating undifferentiated ES cells with DRB. There was no significant difference in the stability of either RNA between wild-type and Xist{Delta}A/Y ES cells.

 

Figure 3
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  		Fig. 3. Xist{Delta}A fails to inactivate the mutated X chromosome. (A) The paternal transmission of Xist{Delta}A results in an extreme bias in the sex ratio of live pups born to wild-type females crossed with X{Delta}AY males in favor of males (upper panel). One of the two females turned out to be XO; therefore, the mutated allele was transmitted to only one female (0.46%). By contrast, the mutated allele was transmitted to both male and female pups at the expected ratio from the mothers (lower panel). (B) Gross morphology of an embryo typical of those that inherited the paternal X{Delta}A (XX{Delta}A). Scale bar: 200 µm. (C) Histological sections of the presumptive XX{Delta}A embryos are shown in comparison with a presumptive male litter mate at E6.5. epc, ectoplacental cone; exe, extraembryonic ectoderm; ee, embryonic ectoderm (D) RT-PCR analysis of allelic expression of X-linked G6pd and Hprt in the trophoblast recovered from E6.5 embryos. XJF1 and X{Delta}A/Xlab are maternal and paternal in origin, respectively. Expression of the paternal copy was evident in XJF1X{Delta}A in both cases. (E) Expression of the GFP transgene on the paternal X in female embryos at E7.5. In contrast to wild-type female embryos (XXGFP), GFP fluorescence was uniformly negative in the embryonic tissues in X{Delta}AXGFP embryos, suggesting that X{Delta}A failed to undergo inactivation even in the embryonic tissues. (F) RT-PCR analysis of the expression of X-linked G6pd and Hprt in the embryonic tissues at E7.5. The maternal copies of both genes on XJF1 were not expressed in X{Delta}AXJF1 embryos, suggesting that X{Delta}A failed to undergo inactivation.

 

Figure 4
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  		Fig. 4. Expression of Xist{Delta}A RNA in preimplantation embryos. RNA-FISH was carried out in XX{Delta}A and XX embryos at the eight-cell and blastocyst stages. At the eight-cell stage, although the accumulation of Xist RNA was evident in XX embryos, expression of Xist{Delta}A RNA in XX{Delta}A was very faint (arrowheads) and subsequently became undetectable by the blastocyst stage. Inset in the panel of the 8-cell XX{Delta}A embryo is an enlargement of the nucleus showing a faint Xist signal (marked with an asterisk).

 

Figure 5
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  		Fig. 5. Xist and Tsix are aberrantly regulated in XX{Delta}A embryos. (A) Quantitative RT-PCR analysis of individual F1 blastocysts recovered from XJF1XJF1 females crossed with either XY or X{Delta}AY males. The level of Xist{Delta}A RNA in XJF1X{Delta}A was much lower than that of wild-type Xist in XX blastocysts. (B) Normally, a silent copy of paternal Tsix was ectopically expressed in XJF1X{Delta}A blastocysts upon paternal transmission of the Xist{Delta}A allele. Following two-round PCR on cDNA prepared from single F1 blastocysts, the amplified products were digested with BsmAI, the recognition site of which is present only in the laboratory strain. (C) Methylation profile of the Xist promoter region on the paternal X in the trophoblast isolated from XX and XX{Delta}A embryos at E6.5 was analyzed by bisulfite sequencing. It was evident that the methylation level was significantly higher in XX{Delta}A than that in XX, suggesting that the expression of normally silent Tsix on the paternal X{Delta}A induces aberrant methylation of the Xist promoter region in cis. An arrow indicates the position of the transcription start site of Xist.

 

Figure 6
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  		Fig. 6. Methylation profile of the Xist promoter region and Xite HS6 in sperm recovered from XY and X{Delta}AY males. (A) Although the Xist promoter region was partially methylated in sperm recovered from XY as previously reported (McDonald et al., 1998Go), it exhibited even lower methylation in the sperm of X{Delta}AY males. (B) The Xite HS6 region, the methylation of which in sperm was suggested to be an imprint responsible for the repression of paternal Tsix in the tissues or cells showing imprinted X-inactivation, was heavily methylated in sperm of both XY and X{Delta}AY males.

 

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