Species differences in TSIX/Tsix reveal the roles of these genes in X-chromosome inactivation

Abstract

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.

Figure 1

Partial maps of (a) human and (b) mouse XIC regions, comparing locations of XIST and TSIX transcripts and of CpG islands. Blackened boxes indicate XIST exons; unblackened boxes indicate CpG islands; and arrows show the transcripts and the direction of transcription. a, Human XIC. The relevant 18–80 kb of the U80460 DNA sequence (GenBank) is shown. h, m, and u are primer sets, and the gray-shaded boxes indicate the FISH probes. b, Mouse XIC. The relevant 60 kb is shown.

Figure 2

Simultaneous hybridization, in situ, of XIST- and TSIX-specific probes to nuclear RNA. In panels a, b, and d, the probes are XIST exon 1 (red) and TSIX-70 (green); overlapping signals (yellow) are seen in merged images. a, TSIX and XIST coexpressed in mouse ES-10 cells carrying the human transgene ( row ES-10), human HED cells (row LVEC), and 46,XX adult human female fibroblasts (row CC). b, Persistence of TSIX transcripts in human female cells from fetus, placenta, and neonate: 5–6 wk gestation (row ET-2), 6.5–8 wk gestation (row Fe18), 2 mo after birth (row TW-2), and full-term placenta, 47,XXX (row PG). c, TSIX expressed from the inactive X chromosome. Sequential TSIX RNA/XIST DNA hybridization showing three XIST alleles (red) but only two TSIX RNA signals (green) in PG (47,XXX) placenta. The arrow indicates the active X chromosome lacking the TSIX signal. d, TSIX expressed from the human inactive X chromosome, of either parental origin, in hydatidiform mole (K1248, with inactive paternal X chromosome) and in clones 1 and 2, from specimen TW-2, that have either the maternal X chromosome (Xi_mat) or the paternal X chromosome (Xi_pat) as the inactive X chromosome, respectively.

Figure 3

Results of RT-PCR analysis, showing persistence of TSIX expression in human female cells of fetal and placental lineages: strand-specific priming of cDNA and amplification with primer set h, for XIST exon 5 (136-bp product) in the presence (+) or absence (−) of reverse transcriptase (RT). Top, Antisense (TSIX). Bottom, Sense (XIST). Specimens are from an adult male (lane 1), an adult female (lanes 2 and 3), mouse ES-10 cells with the transgene (lanes 4 and 5), a female HED cell, LVEC (lanes 6 and 7), placentas CV2 (lanes 8 and 9) and CV5 (lanes 10 and 11), and fetal fibroblasts at 12 wk (lanes 12 and 13) and at 18 wk (lane 14). Extra bands for ES-10 are of mouse origin.