Abnormal X: autosome ratio, but normal X chromosome inactivation in human triploid cultures

Abstract

Background: X chromosome inactivation (XCI) is that aspect of mammalian dosage compensation that brings about equivalence of X-linked gene expression between females and males by inactivating one of the two X chromosomes (Xi) in normal female cells, leaving them with a single active X (Xa) as in male cells. In cells with more than two X's, but a diploid autosomal complement, all X's but one, Xa, are inactivated. This phenomenon is commonly thought to suggest 1) that normal development requires a ratio of one Xa per diploid autosomal set, and 2) that an early event in XCI is the marking of one X to be active, with remaining X's becoming inactivated by default.

Results: Triploids provide a test of these ideas because the ratio of one Xa per diploid autosomal set cannot be achieved, yet this abnormal ratio should not necessarily affect the one-Xa choice mechanism for XCI. Previous studies of XCI patterns in murine triploids support the single-Xa model, but human triploids mostly have two-Xa cells, whether they are XXX or XXY. The XCI patterns we observe in fibroblast cultures from different XXX human triploids suggest that the two-Xa pattern of XCI is selected for, and may have resulted from rare segregation errors or Xi reactivation.

Conclusion: The initial X inactivation pattern in human triploids, therefore, is likely to resemble the pattern that predominates in murine triploids, i.e., a single Xa, with the remaining X's inactive. Furthermore, our studies of XIST RNA accumulation and promoter methylation suggest that the basic features of XCI are normal in triploids despite the abnormal X:autosome ratio.

Figure 1

Counting Xi's and X's in control and triploid fibroblasts. A. XIST RNA FISH was used to determine Xi content in cells. In this example, two cells from an early passage of 75-29 XXX triploid fibroblasts are shown, one with a single XIST RNA body, and the other with two (arrows). B. X chromosome content was determined using FISH to X-linked alpha centromeric DNA (CEP X probe). In this example, cells from a clone of 75-29 (F10) all show three X-centromere signals.

Figure 2

XIST RNA signal intensity distribution in control and triploid cells. Fluorescence intensities were quantified for XIST RNA FISH signals in normal diploid female fibroblasts (81-58), GM04939 XXX triploids with a single XIST signal, and GM04939 XXX triploids with two XIST signals (see "Methods"). For GM04939 cells with two XIST signals, the intensities of each signal are shown separately and in sum.

Figure 3

G6PD promoter methylation in control and triploid cells. A. Map of the 457 bp G6PD promoter region containing the CpG sites we analyzed for methylation (numbered 1–52). B. Methylation patterns from an early passage culture of GM04939 XXX triploid fibroblasts. CpG sites are represented as either methylated (■) or unmethylated (□); a question mark indicates that methylation status could not be determined. Each clone represents the methylation pattern of a single chromosome, and the number of methylated CpG's in each clone is given in the right column; clones are ordered from lowest to highest methylation. C. Methylation patterns from a late-passage culture of GM04939 XXX triploid fibroblasts. The proportion of hypermethylated clones (Xi alleles) is decreased in later passage cells, in agreement with the decrease in the number of Xi's seen cytologically. D. Methylation patterns from a culture of GM04376 XXX triploid fibroblasts that has a preponderance of cells with one Xi; the methylation patterns we observed are consistent with this deficit in Xi's relative to Xa's.