Xist yeast artificial chromosome transgenes function as X-inactivation centers only in multicopy arrays and not as single copies

Abstract

X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.

FIG. 1

(A and B) Structures of the two YACs used in this study, PA-2 (460 kb) and PA-3 F1n (320 kb). More detailed physical maps of these YACs have been published previously (20, 23). The DNA probes used in characterization of the YACs and transgenic clones are shown as black boxes below each YAC map along with the I-PpoI, SalI (Sa), and SfiI (Sf) sites that were informative in the analysis of the transgenes. (A) Transgene structures of the L4 (lipofection-derived) and F1 (spheroplast fusion-derived) lines created with the 460-kb YAC PA-2. I-PpoI meganuclease sites present at each extremity of this YAC enabled YAC integrity of the transgenic clones to be assessed (see panel C). SfiI mapping (data not shown) provided further resolution concerning the extent of the region covered by the transgenes (particularly in line F1 A). Multicopy transgenic line F1 C showed, in addition to the major YAC fragment illustrated, a minority of smaller transgenic fragments. (B) The transgene structures of L1 (lipofection-derived) lines created with the 320-kb YAC PA-3 F1n. Both SalI and SfiI restriction sites were used to analyze the structures of these transgenes (see panel D for an example of the SfiI analysis). A SalI site within the YAC arm of PA-3 F1n which leads to the generation of a SalI band detected by the Tsx and DXPas34 probes of approximately the same size as that at the endogenous locus is indicated as Sa*. It should be noted that there are other SalI sites present in this region (23), but these are methylated and thus uncut by SalI in mouse genomic DNA. Multicopy transgenic line L1 17 showed, in addition to the major YAC fragment illustrated, a minority of smaller transgenic fragments. (C) Example of pulsed-field gel analysis of I-PpoI-digested DNA of cell lines carrying YAC PA-2 transgenes and the nontransgenic control lines CK35 and HP310. In lines L4 8, L4 13, and F1 E (single-copy transgenes), a 460-kb fragment detected by all probes tested lying within the YAC (Xist probe shown) following I-PpoI digestion suggested that the YAC was intact. In lines F1 A (single copy) and L4 12 (two copy), larger (>750-kb) transgene fragments were detected, suggesting that at least one I-PpoI site had been lost. In line F1 C (three to four copies), detection of a 360-kb band as well as a >750-kb band suggested that at least two copies of the truncated YAC, as shown in panel A, were present, with additional end fragment(s) (i.e., minus a second I-PpoI site) being responsible for the higher-molecular-weight band. (D) Example of pulsed-field gel analysis of SfiI-digested DNA of lines carrying YAC PA-3 F1n transgenes. The internal SfiI bands detected by various probes (B) appear to be intact in lines L1 25 (single copy), L1 6 (two copies) and L1 12 (six to seven copies) and are detected as an increase in the intensity of the endogenous bands compared with the control cell line CK35 (170 and 280 kb with the Xist probe shown here). In L1 17 (five to eight copies), the majority of the SfiI fragments appear to be intact, although a minority of smaller and larger fragments, corresponding to truncated fragments and partial digests of neighboring transgene copies, respectively, can also be seen.

FIG. 2

Xist RNA expression in single-copy and multicopy transgenic lines in undifferentiated ES cells. (A) DNA FISH on metaphase chromosomes from line L1 17 showing a single integration site for the multicopy transgene in a mouse autosome. A spectrum red-labeled YAC PA-3 F1n probe hybridized with both the transgenic locus (T) and its endogenous site on the X chromosome (X). The larger signal at the transgene locus reflected its higher copy number (seven in this case). This was confirmed in other hybridizations involving a YAC-specific probe. (B to F) Under conditions that do not denature chromosomal DNA, undifferentiated ES cells were hybridized with a spectrum green labeled Xist probe to detect Xist RNA. In lines L4 8 (single copy, XY) (B), F1 A (single copy, XX) (C), and 53.2 (two unlinked copies, XO) (D), Xist RNA pinpoint signals over the X chromosome and the single-copy transgene loci were indistinguishable. In lines L4 12 (multicopy, XY) (E), and L1 17 (multicopy, XY) (F), the transgenic Xist RNA pinpoint tended to be slightly larger than the endogenous signal. (D and F) The Xist RNA FISH signals in positioned nuclei were photographed, and the cells were then denatured and DNA FISH was performed. (D) A probe specific for the YAC vector (pYAC4, spectrum red labeled) detected the two single-copy transgenes (T) in line 53.2; (F) a probe specific for the X chromosome (BAC X, spectrum red labeled) demonstrated that the larger Xist pinpoint signal corresponded to the seven-copy transgene (T) present in line L1 17. (G to I) Two-color RNA FISH for simultaneous detection of Xist (spectrum green) and Brx (spectrum red) transcripts under nondenaturing conditions (see above). (G) L4 8 (single copy, XY); (H) F1 A (single copy, XX); (I) L4 12 (two copy, XY).

FIG. 3

Xist and Brx expression in single-copy and multicopy transgenic lines in fully differentiated ES cells. (A to D) Under conditions that do not denature chromosomal DNA, ES cells differentiated by EB attachment and outgrowth on chamber slides for several days, were hybridized with a spectrum green-labeled Xist probe to detect Xist RNA as well as a spectrum red-labeled Brx probe to detect Brx RNA in panel D. (A) Several nuclei in the control female cell line (HP310, XX) showing Xist RNA domains (inactive X chromosomes) in the majority of cells; in some cells, a single Xist RNA pinpoint (on the active X chromosome) is also detected. (B) In differentiated cells of the control male cell line (CK35, XY), either a single Xist RNA pinpoint or no Xist signal at all is detected. (C) In the single-copy transgenic line L4 8, either one, two, or no Xist RNA pinpoints are detected. (D) Two representative differentiated nuclei of line 53.2 (two unlinked single-copy transgenes, X0) are shown, one with no Xist RNA signals (green) but three Brx signals (red) and the other with three Brx signals and a single Xist RNA signal (yellow as it overlaps with Brx). (E and F) Line F1 A (single-copy transgene, XX) simultaneous RNA-DNA two-color FISH on mildly denatured nuclei (see Materials and Methods), using a spectrum green-labeled Xist probe and spectrum red-labeled YAC vector (E) or BAC X (F) probe. The Xist RNA domain is associated with the X chromosome (X) and not the single-copy transgene (T). (G to I) RNA FISH on undenatured nuclei of differentiated L4 12 ES cells (multicopy, XY) using a Xist probe (spectrum green) was followed by denaturation and DNA FISH using a spectrum red-labeled YAC vector probe. In the majority of nuclei (G), a single Xist RNA domain associated with the transgene (T) was observed. Occasionally the Xist RNA domain was associated with the X chromosome (H) or with both the X chromosome and the transgene (I).

FIG. 4

Evolution of Xist RNA signals in differentiating ES cells. (A) One-day EB of control female ES line HP310; two-color RNA FISH detecting Xist (spectrum green) and Brx (spectrum red) transcripts. Xist pinpoint clusters can sometimes be seen at this stage. We considered “clusters” to consist of 2 to 10 distinct pinpoints randomly scattered around the site of transcription, while “domains” were dense, more extensive signals that covered an interphase chromosome-sized area. (B) Two-day EB, control female ES line HP310; Xist (spectrum green) RNA FISH showing a Xist RNA domain. (C) One day EB of line L4 8 (single-copy transgene, XY); two-color RNA FISH detecting Xist (spectrum green) and Brx (spectrum red) transcripts. Xist pinpoint clusters can sometimes be seen at this stage. (D) L4 8 1-day EB. Performance of Xist RNA FISH (spectrum green) was followed by denaturation and DNA FISH using a YAC-specific probe (spectrum red), which shows that Xist RNA pinpoint clusters tend to be associated with the transgene loci. (E and F) L4 12 (two-copy transgene, XY), 2-day EB. Performance of Xist RNA FISH (spectrum green) was followed by denaturation and DNA FISH using a YAC-specific probe (spectrum red). (E) Xist RNA pinpoint clusters are sometimes seen either at the transgenic locus alone or (as shown here) at both the X chromosome and the transgene (T). (F) Xist RNA domains are mostly found to be associated with the transgene. (G) Representative view of L4 12 cells (two-copy transgene, XY) from 1-day EBs. Two-color RNA FISH detecting Xist (spectrum green) and Brx (spectrum red) transcripts shows that the majority of cells contain one or two Xist pinpoint clusters.

FIG. 5

Mosaicism in the presence of Xist RNA domains in fully differentiated ES cells. (A) Xist RNA FISH (spectrum green) on differentiated L1 17 cells (five- to eight-copy transgene, XY). Shown is a representative view with Xist RNA domains present in the majority of cells. (B) Xist RNA FISH (spectrum green) on differentiated L4 12 cells (two-copy transgene, XY). Shown is a representative view with Xist RNA domains present in a minority of cells. (C) Two-color RNA FISH detecting Xist (spectrum green) and Brx (spectrum red) on differentiated L412 cells (two-copy transgene, XY) as in panel B. In cells containing a Xist RNA domain, no Brx signal is detected within the domain (indicating Brx inactivation), while a Brx signal is detected on the other (active) Xic in these cells. In cells with no Xist domain, two Brx signals are detected, indicating that despite the lack of Xist expression and domains in these cells, the transgene is still functional (i.e., Brx is expressed). In all cases, ES cells were differentiated for a total of 9 days (4 days of EB growth in suspension followed by 5 days of outgrowth after EB attachment onto chamber slides) and fixed in situ on the slides.