A new Xist allele driven by a constitutively active promoter is dominated by Xist locus environment and exhibits the parent-of-origin effects

Abstract

The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist CAG ) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist CAG resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist CAG in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4-to 8-cell stages when paternally inherited, and Xist CAG was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.

Defects in dosage compensation impact global gene regulation in the mouse trophoblast

“…This suggests that a mechanism for the random choice of monoallelic upregulation is still effective on either the wild-type or Xist CAGΔ5′ allele despite the fact that Xist CAGΔ5′ is driven by the CAG promoter. This appears consistent with our recent finding that, in undifferentiated ESCs, the CAG promoter driving the Xist CAG allele does not efficiently promote transcription but rather behaves in a manner similar to the endogenous Xist promoter, causing only a basal level of transcription (Amakawa et al, 2015). We anticipate that, given the constitutively active nature of the CAG promoter in many differentiated cells, monoallelic upregulation of the wild-type Xist allele at the onset of XCI should be followed by upregulation of Xist An X chromosome acquires a series of epigenetic modifications following the accumulation of Xist RNA and forms a silencing compartment, into which X-linked genes that are to be silenced relocate (Escamilla-Del-Arenal et al, 2011;Patrat et al, 2009).…”

“…Since our previous study revealed that a simple deletion of the A-repeat abolished Xist upregulation during embryonic development (Hoki et al, 2009), we decided instead to express Xist RNA lacking the A-repeat using a CAG promoter, using the same approach as we used previously to create the Xist CAG allele (Amakawa et al, 2015). The 5′ region of Xist spanning from its endogenous promoter to the XhoI site 0.9 kb downstream of the major transcription start site in exon 1 was replaced with a fragment containing the CAG promoter and a floxed selection marker by gene targeting to generate the Xist CAGΔ5′-2L allele (Fig.…”

“…Since Xist RNA is exclusively expressed from the Xist CAG allele in the embryonic tissue of XX CAG (Amakawa et al, 2015), we expected that only the Xist CAGΔ5′ allele would be upregulated in XX CAGΔ5′ embryonic tissue. This was not the case, however, and the wild-type Xist allele was also upregulated in addition to the mutated Xist CAGΔ5′ allele, implying that the wild-type Xist allele was secondarily upregulated so as to achieve dosage compensation in XX CAGΔ5′ embryos, in which Xist CAGΔ5′ RNA failed to induce a sufficient level of XCI.…”

“…In a previous study, we found that all +/Xist CAG embryos obtained from a cross between wild-type females and Xist CAG2L /Y; Tg(Pgk2-cre) males were apparently normal and expressed the full-length Xist RNA under control of the CAG promoter (Amakawa et al, 2015). It was therefore reasonable to expect that Xist CAGΔ5′ RNA, although lacking the A-repeat, would be expressed and thus we would be able to evaluate its silencing function.…”

“…A targeting vector was constructed by modifying pCAG-CΔM20, which was previously constructed to generate the Xist CAG2L allele (Amakawa et al, 2015). pCAG-CΔM20 was digested with SalI and XhoI to release a 3.7 kb CAG-Pac cassette and a 0.9 kb Xist 5′ fragment (nt 21-912).…”