Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

<div><p>X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent <i>in situ</i> hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.</p></div

Frequency of nuclei transcribing both alleles of neighbouring loci.

<p>Expected frequencies of nuclei transcribing from both allelels of neighbouring loci were calculated from observed frequencies of 2Z-active (or 2X-active) nuclei (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635.s006" target="_blank">Table S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635.s007" target="_blank">S2</a>). For example, <i>SMARCA2</i> was 2Z-active in 76% of nuclei and <i>PTPRD</i> was 2Z-active in 60% of nuclei. Therefore, it was expected that they should both be 2Z-active in 0.76*0.6 (45.6%) of nuclei. P-values were calculated with a X² test with 1 degree of freedom. Bonferroni correction was conducted.</p

RNA-FISH activity maps of the platypus Xs and chicken Z chromosomes.

<p>Bars represent the percentage of homogametic nuclei transcribing 2 (blue), 1 (red) or 0 (grey) alleles for each locus. Loci in pseudoautosomal regions (grey boxes) were tested in both male (indicated by a circle) and female (indicated by a star). Green coloring on platypus Xs represents homology to the chicken Z <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635-Veyrunes1" target="_blank">[23]</a>. Platypus X chromosomes are not to scale (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635.s005" target="_blank">Figure S5</a>). Genes denoted by * were analysed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635-Deakin1" target="_blank">[24]</a>.</p

Transcriptional activity of neighbouring chicken Z loci and platypus X₅ loci in fibroblasts.

<p>Gene names are color coded to correspond to signal color. <b>A</b>) In nuclei with only one allele active for both genes the signals co-locate in both sexes. <b>B</b>) Nuclei from the homogametic sex in which both genes are 2Z/2X-active. <b>C</b>) Nuclei from the homogametic sex in which the active Z/X expresses both genes and the other (inactivatable) Z/X expresses only one gene.</p

RNA-FISH analysis of transcription from neighbouring loci in homogametic nuclei.

<p>RNA-FISH analysis of transcription from neighbouring loci in homogametic nuclei.</p

Proposed mechanism of telomere length regulation in the dasyurid germline.

<p>Primordial germ cells contain a mixture of long and short telomeres which would be expected to segregate randomly, followed by lengthening and diminution in the paternal and maternal germlines, respectively. Following fertilisation, the zygote contains haploid chromosome sets with long and short telomeres which are maintained in somatic cells.</p

Phylogenetic tree of the species studied here.

<p>Telomere length dimorphism is a feature of the Family Dasyuridae and arose after the divergence of the Orders Dasyuromorphia and Paramelemorphia, up to 50 million years ago.</p

Telomere length dimorphism in the Tasmanian devil.

<p>Q-FISH of metaphases from a male and a female Tasmanian devil (lymphocytes and skin fibroblasts, respectively) and a DFTD cell-line using a Cy3-labelled (CCCTAA)₃ PNA probe. (A) In both male and female Tasmanian devils, homologous chromosomes are characterised by striking differences in telomere length. In male devils, telomeres on the Y chromosome are consistently long and X chromosome telomeres are short. DFTD telomeres are uniformly short. (B) Frequencies of male, female and DFTD telomere fluorescence intensities demonstrate an unusual bimodal distribution of long and short telomeres in male and female devils with marked heterogeneity of the long telomere subset. Telomere lengths are compared to those of C57BL/6 mouse fibroblasts.</p

TERT expression and telomerase activity in DFTD and Tasmanian devil tissues.

<p>(A) RT-PCR using TERT primers was performed on DNA from Tasmanian devil testis (Te), spleen (Spl) and lymph node (LN) and four independent DFTD tumours (T1–4). GAPDH RT-PCR was performed on DNA from the same tissues as a loading control. RT, reverse transcriptase. (B) Telomerase activity is detected in three DFTD tumors (T1–3) and two testis samples (Te1 and Te2), as measured by TRAP assay.</p

Distantly related marsupials have uniform telomeres.

<p>Marsupials in the order Diprotodontia, including the Tammar wallaby (<i>Macropus eugenii</i>), the common wombat (<i>Vombatus ursinus</i>), the common brushtail possum (<i>Trichosurus vulpecula</i>) and the Rufous bettong (<i>Aepyprymnus rufescens</i>), and in the order Paramelemorphia, represented by the eastern barred bandicoot (<i>Perameles gunnii</i>), have uniform telomeres between homologous chromosomes. The short beaked echidna (<i>Tachyglossus aculeatus</i>), a monotreme mammal, likewise has uniform telomeres.</p