A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation - PubMed (original) (raw)

A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation

Y K Hong et al. Proc Natl Acad Sci U S A. 1999.

Abstract

In this report, we present structural data for the murine Xist gene. The data presented in this paper demonstrate that the murine Xist transcript is at least 17.4 kb, not 14.3 kb as previously reported. The new structure of the murine Xist gene described herein has seven exons, not six. Exon VII encodes an additional 3.1 kb of information at the 3' end. Exon VII contains seven possible sites for polyadenylation; four of these sites are located in the newly discovered 3' end. Consequently, it is possible that several distinct transcripts may be produced through differential polyadenylation of a primary transcript. Alternative use of polyadenylation signals could result in size changes for exon VII. Two major species of Xist are detectable by Northern analysis, consistent with differential polyadenylation. In this paper, we propose a model for the role of the Xist 3' end in the process of X-chromosome counting and choice during embryonic development.

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Figures

Figure 1

The murine Xist gene consists of seven exons. (A) Revised genomic structure of Xist contains a 781-bp-long intron compared with the documented structure of exon VI by Brockdorff et al. (14). Exon VI/exon VII junction sequences are shown. Arrowhead, _Ava_I; asterisk, _Eco_RI. (B) Comparison of _Ava_I/_Eco_RI fragments from mouse genomic DNA, Y116 (lane 2), and female mouse lung cDNA (lane 3). PCR fragment spanning the region was cloned and sequenced. Lane 1 is 1-kb DNA size marker (GIBCO/BRL).

Figure 2

New structure of the 3′end Xist. (A) The four ESTs (EST1≈4) are mapped relative to exon VII of Xist (GenBank accession nos. of EST1 ≈ 4: AA543875, AA221611, AA690387§, and R74734, respectively). PCR fragments synthesized by using primers whose locations are marked by closed arrowheads are shown to demonstrate the colinearity of all the ESTs. Locations of primers used to determine the end of the Xist transcript are marked by open arrowheads. Probes used for Northern blots and RNA–FISH (pWS854, pWS850) are also indicated. Consensus sequences for polyadenylation (A_n_) and sequences of putative stem and loop structures are also localized. The 65-kb deletion created by Clerc and Avner (12) begins from the _Sca_I site (marked with heavy arrow) in the EST1. EST fragments were recovered as described in Experimental Procedures. (B) All the ESTs are colinear with Xist. All PCR products were sequenced. For the purpose of this figure, the PCR fragments for Xist + EST1 and for EST1 + EST2, EST2 + EST3, and EST3 + EST4 were electrophoresed in 0.8% and 2% agarose gels, respectively, transferred to nylon membranes, and hybridized with individual fragments (probes for this figure, Xist, EST1, EST2, and EST3, respectively). (C) YAC116 (genomic DNA); ♀, female mouse lung cDNA library; ♂, male mouse brain and male heart cDNA libraries. Approximate DNA sizes are marked by using either 1-kb marker (GIBCO/BRL) for 0.8% gel or 100-bp marker (NEB, Beverly, MA) for 2% gel. The top of each lane is the origin of migration. § Note: Accession no. AA690387 is incorrectly identified as derived from a male mouse cDNA library in GenBank. It is correctly attributed to a female library on the I.M.A.G.E. home page (

http://www-bio.llnl.gov/bbrp/image/image.html

Figure 3

Northern blot showing major and minor Xist species. Murine male (♀) and female (♂) kidney RNA was fractionated on a formaldehyde-agarose gel and transferred to positively charged nylon yielding duplicate strips. Duplicate lanes were hybridized to the individual probes: pWS850, pWS854, and mx8 (20). The lane containing mx8 is not shown, because there was no hybridization signal. Xist transcripts, both major and minor, are indicated by arrow symbols. Positions of ribosomal RNA are indicated to give an indication of relative mobility. The figure shows two major species of Xist using the pWS850 probe. The new 3′-end probe, pWS854, hybridizes disproportionately to the larger of the two major species of Xist RNA.

Figure 4

RNA–FISH photomicrograph of male and female somatic cells. RNA–FISH was performed to visualize cytoplasmic/nuclear RNA that hybridized to Xist probes pWS850 (FITC) and pWS854 (rhodamine). Hybridizations of Xist probes were performed simultaneously and separate channels recorded; they were merged after recording. Micrograph shows the colocalization of the probes pWS850 and pWS854. (×600.)

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A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation - PubMed (original) (raw)