Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells - PubMed (original) (raw)

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells

E Heard et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

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Figures

Figure 1

Structural analysis of human XIST transgenes. (a) Human YAC 19C12 is 480 kb long (24). XIST is flanked by about 380 kb of 5′ and 70 kb of 3′ sequence. The XIST gene and LAMRP4 pseudogene are shown. The orientation of XIST transcription is indicated with an arrow. Meganuclease I-_Ppo_1 sites were introduced into both YAC vector arms to facilitate assessment of YAC integrity in transgenic cells (25) along with the _pgk-neo_-selectable marker, introduced into one arm, to enable selection for YAC uptake after lipofection into CK35 male ES cells. (b) _Eco_RI-digested DNA of control CK35 ES cells and transgenic clones hY1, hY5, hY20, and hY21 hybridized with probes for: LYS2, a yeast marker present in one of the YAC vector arms (Top); human XIST (6/7r in exon 1) (Middle); and mouse Xist (HF) (Bottom). Using these probes and others, transgene copy number was estimated to be 1–2 for lines hY1 and hY5, 8–10 for line hY20, and 10–14 for hY21. (c) I-_Ppo_1-digested DNA of transgenic clones hY1, hY5, hY20, and hY21 resolved on a pulse-field gel. Hybridization with either XIST (shown here), LAMRP4, or YAC vector probes revealed a 480-kb band corresponding to intact YAC sequences in lines hY1, hY5, hY20, and hY21. Lanes corresponding to these clones, from the same autoradiograph, have been juxtaposed. The band in hY5 appears slightly lower in size as a result of aberrant migration because of DNA-loading differences between lanes (as judged by ethidium bromide staining). In line hY20, the additional smaller fragment suggests the presence of some rearrangement in a subpopulation of the transgenic sequences.

Figure 2

RT-PCR analysis of human XIST expression in transgenic mouse ES cells. (a) The human XIST gene is shown with the locations and orientations of the six primer pairs (–6) used (see Materials and Methods). The previously determined splicing pattern (7) is shown, with less frequently observed splicing events indicated by dashed lines. (b) Amplification of cDNA in transgenic cells before and after differentiation (8-day EBs). Positive control human cDNA is female brain. Mouse controls are the host ES cell line CK35 and female kidney. Lanes: 1, hY20 undifferentiated; 2, hY20 EBs; 3, hY21 undifferentiated; 4, hY21 EBs; 5, hY1 undifferentiated; 6, hY1 EBs; 7, hY5 undifferentiated; 8, hY5 EBs; 9, CK35 undifferentiated; 10, human female brain; 11, mouse female kidney. Representative data using primer pairs 3 and 6 are outlined in a. Mouse Hprt RT-PCR was used to control the RT samples. The PCR products shown are cDNA-specific as the primers flank introns.

Figure 3

FISH analysis of human and murine Xist expression in transgenic mouse ES cells. RNA FISH on undenatured nuclei and DNA FISH after chromatin denaturation (see Materials and Methods). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). (a) Human XIST RNA (green) detected in human amniocytes: a domain-like signal over the human X chromosome (8). (b) Representative example of human XIST RNA domain (green) and mouse Xist RNA pinpoint (red) signals in undifferentiated ES cells of line hY20 or hY21. (c) Human XIST RNA domain (green) in line hY21 after differentiation (8 days). (d) Human XIST RNA (red) and chromosome 2 DNA (green) in transgenic hY20 cells; overlapping signals appear yellow. (e) Multiple differentiated cells of transgenic line hY21, illustrating the degree of localization of the human XIST RNA (red) over chromosome 13 (green), which was high in the majority of cells and more restricted (e.g., top left) or dispersed (e.g., bottom left) in a minority of cells. Similar observations were made in undifferentiated hY21 cells. Overlapping signals appear yellow. (f) Human XIST RNA (green) and Dhfr/Rep-3 RNA (red) signals detected in undifferentiated hY21 nuclei. (g) Human XIST RNA (green) and Dhfr/Rep-3 RNA (red) signals detected in differentiated (10-day) hY21 cells. (h) Human XIST (red) and mouse Xist (green) RNA domains detected in differentiated (10-day) transgenic hY21 cells.

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Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells - PubMed (original) (raw)