Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

Abstract

Background: X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue.

Results: We examined expression of X-borne genes using quantitative PCR, revealing a range of dosage compensation for different loci. To assess the frequency of 1X- or 2X-active fibroblasts, we investigated expression of 32 X-borne genes at the cellular level using RNA-FISH. In female fibroblasts, two-color RNA-FISH showed that genes were coordinately expressed from the same X (active X) in nuclei in which both loci were inactivated. However, loci on the other X escape inactivation independently, with each locus showing a characteristic frequency of 1X-active and 2X-active nuclei, equivalent to stochastic escape. We constructed an activity map of the tammar wallaby inactive X chromosome, which identified no relationship between gene location and extent of inactivation, nor any correlation with the presence or absence of a Y-borne paralog.

Conclusions: In the tammar wallaby, one X (presumed to be maternal) is expressed in all cells, but genes on the other (paternal) X escape inactivation independently and at characteristic frequencies. The paternal and incomplete X chromosome inactivation in marsupials, with stochastic escape, appears to be quite distinct from the X chromosome inactivation process in eutherians. We find no evidence for a polar spread of inactivation from an X inactivation center.

Figure 1

Female:male ratio for average expression of tammar X-borne genes in fibroblast cells (five males, six females) normalized to the autosomal GAPDH housekeeping gene. Genes are presented in the order in which they are located on the X, from the centromere down. Ratios varied between complete compensation (ratio 1.0) and no compensation (ratio 2.0). *, statistically significant association (P < 0.05).

Figure 2

Physical map of the tammar wallaby X chromosome showing location of analyzed genes. Locations of BACs and fosmids used for RNA-FISH on the tammar X chromosome. The DAPI dense regions are indicated in grey. BAC and fosmid clones used in this study and the genes they bear, genome coordinates and the band location of human orthologues are shown.

Figure 3

Transcriptional activity of an X-borne gene and autosomal control in male fibroblasts. Loci are color coded above panels. (a) Male fibroblast nuclei with transcription from two autosomal GBA alleles (green) and the single X-borne locus (red). (b) Analysis of ATRX by sequential RNA-DNA FISH. Merged panel reveals that the RNA (red) and DNA (green) FISH signals co-localize with no cross-hybridization to the Y paralogue. Nuclei are counterstained with DAPI (blue).

Figure 4

Coordinate transcriptional activity of neighboring X-borne loci assayed by two-color RNA-FISH in male and female fibroblasts. Loci are color coded above panels. (a) Male nuclei with transcription from two X-borne loci on the single X chromosome. (b) Female nuclei with transcription from two X-borne loci on the active, but not the inactive, X chromosome. Nuclei are counterstained with DAPI (blue).

Figure 5

Transcriptional activity of an X-borne gene and autosomal control in female fibroblasts. LRCH2 (red signal) is on the X and GBA (green signal) is on chromosome 2. (a,b) Female fibroblast nucleus shows transcription from both autosomal GBA alleles (green), and either one (a) or two (b) X-borne LRCH2 alleles (red). Nuclei are counterstained with DAPI (blue).

Figure 6

Expression and localization of ATRX by RNA-DNA FISH in female fibroblast nuclei. (a,b) Sequential ATRX RNA (red) and DNA (green) FISH reveals that either one (a) or two (b) RNA-FISH signals co-localize with the DNA signals. Nuclei are counterstained with DAPI (blue).

Figure 7

X chromosome activity map in tammar wallaby female fibroblasts. RNA-FISH activity map of the tammar wallaby X chromosome. Bars represent percentage of nuclei transcribing from 2 (blue), 1 (red) or 0 (grey) loci. The absence of polarity suggests that no inactivation center co-ordinates inactivation. *X genes with known Y paralogues.

Figure 8

Transcriptional activity of an X-borne gene and its Y paralogue in male and female fibroblasts. The HUWE1Y probe (red signal) detects the paralogue located on the Y, and the HUWE1X probe (green signal) detects the paralogue on the X chromosome. (a) Male nucleus with transcription from the single X locus (HUWE1X, green) and the single Y locus (HUWE1Y, red). Different signal intensities from different probes does not correlate to transcription level. (b) Female fibroblast nuclei with transcription from one (left) and two (right) X-borne loci (HUWE1X, green), and no expression detected with the Y-specific probe (HUWE1XY, red). Nuclei are counterstained with DAPI (blue).

Figure 9

Two-color RNA-FISH in female fibroblasts reveals independent escape from inactivity of two neighboring X-borne loci. Loci are color coded above panels. (a) Nuclei in which one gene (green) is expressed from both alleles and the second gene (red) is expressed from only one allele. (b) Nuclei in which one gene (green) is expressed from one allele and the second gene (red) is expressed from both alleles. (c) Nuclei in which both genes are expressed from both alleles. ND, no nuclei were observed in this category. Nuclei are counterstained with DAPI (blue).