Further examination of the Xist promoter-switch hypothesis in X inactivation: evidence against the existence and function of a P(0) promoter

Abstract

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P(0)) produces an unstable transcript, while the known downstream promoter (P(1)) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P(0) but retaining P(1). We confirm the previous finding that P(0) is dispensable for Xist expression in undifferentiated cells and that P(1) can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P(1) is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P(0) does not represent the 5' end of a distinct Xist isoform. Instead, P(0) is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P(1) originates from the DNA strand opposite Xist and represents the 3' end of the antisense Tsix RNA. Thus, these data do not support the existence of a P(0) promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

Figure 1

P₀ is not required for Xist expression in transgenic ES cells. (A) RNA fluorescence in situ hybridization (FISH) was performed with sense-specific exon 1 and 6 Xist probes (green). Nuclei shown are from πJL3.9 ES cells, but πJL3.1 yielded identical results. (B) RACE (5′) showed that transgenic cells initiated Xist expression at P₁ and two other potential start sites at approximately −200 bp and +200 bp relative to P₁. RACE (5′) results were obtained from πJL3.9. Note that, because transgenic cells also carried the endogenous Xist gene, detected transcripts may reflect either endogenous or transgenic Xist expression.

Figure 2

Transcription from the P₁ promoter does not yield intrinsically stable RNA. (A) Male, female, and transgenic ES cells or fibroblasts (somatic) were treated with 5 μg/ml actinomycin D for 0, 2, and 4 h and then subjected to RNA FISH with strand-specific pooled exon 1 and exon 6 Xist probes. For each sample, 100 nuclei were scored for the presence of Xist signals at every time point. Because the female ES line is a mosaic of 40XX and 39XO cells, either one or two Xist signals were scored as positive. (B) RNA FISH on metaphase chromosomes of transgenic cells showed that Xist RNA did not coat the chromosome in cis either before (shown) or after differentiation (data not shown). (Inset) Transgenic cell line πJL2.5 (17), a control showing that Xist RNA could coat the autosome on cell differentiation when expressed from a transgene containing 80 kb of Xic sequence.

Figure 3

Identification of a Rps12 pseudogene, pS12X. (A) Alignment of the longest expressed sequence tag corresponding to mouse ribosomal protein S12 cDNA (GenBank accession no. AI526798) and pS12X (bp 3,015–2,472 of GenBank accession no. AJ010350) sequences. Direct repeats (DR) are in grey boxes. Poly(A) stretch is found immediately before the 3′ direct repeat. CJ10–CJ12 primer locations are indicated by arrows. CJ9 is located outside of known Rps12 expressed sequence tags and cDNA sequence. (B) Tissue distribution of the CJ11–CJ12 RT-PCR product. Total RNA from male (M) or female (F) ES cells, adult fibroblasts (Fib), brain (B), and liver (L) was reverse transcribed by random priming and amplified with primers CJ11 and CJ12. −RT controls were processed in parallel without adding RT. (C) Northern blot analysis of total ES and fibroblast RNA with a pS12X probe. ES cells are male (J1, 40XY), female (EL16, a mosaic cell line: 30% 40XX and 70% 39XO), and transgenic (Tg, 116.6).

Figure 4

The RNA detected by CJ11–CJ12 is found in the cytoplasm and is exclusively derived from autosomal Rps12 expression. (A) Nuclear and cytoplasmic distribution of the “P₀ RNA” isolated from male (M) and female (F) fibroblasts (Fib) and ES cells, RNA was reverse transcribed and amplified with CJ11 and CJ12. Xist was amplified with primers Mix20–Mx23b (12), which spans exons 3 to 6 of Xist. (B) TaqI and HinfI restriction maps for pS12X and Rps12 fragments bounded by CJ11 and CJ12. Sizes are shown for polymorphic fragments. Asterisks indicate RFLP positions. (C) RFLP analysis of CJ11–CJ12 RT-PCR products. PCR products were diluted and extended one cycle to minimize heteroduplex formation and then digested with TaqI or HinfI. Polymorphic restriction fragments were detected by hybridization to radiolabeled nested oligonucleotide CJ10. + and − indicate the presence or absence, respectively, of restriction enzyme during incubation. (D) Sensitivity of the RFLP assay of CJ11–CJ12 amplification. A constant amount of Rps12 RT-PCR product was mixed with 10-fold dilutions of pS12X PCR product, digested with TaqI, and visualized by hybridization to CJ10 oligonucleotide. pS12X fragments were visible at 10⁻³ dilution (shown) and at 10⁻⁴ dilution on the original autoradiogram (data not shown).

Figure 5

Map of the region upstream of the P₁ promoter and positions of primers used. (A) A map of the 7-kb region upstream of Xist. The positions of the previously described pseudogene pS19X (27), the Tsix and Xist genes, and the newly identified pseudogene, pS12X, are shown. The precise 3′ end of Tsix has not been defined (denoted by dotted line). Each asterisk designates a sense and antisense primer pair (see Material and Methods). (B) Strand-specific RT-PCR at positions 1–7 and at Rrm2. Identical results were obtained from male, female, and transgenic ES cells. s, sense (Xist strand); as, antisense (Tsix strand).