A cross-species comparison of escape from X inactivation in Eutheria: implications for evolution of X chromosome inactivation

Abstract

Sex chromosome dosage compensation in both eutherian and marsupial mammals is achieved by X chromosome inactivation (XCI)--transcriptional repression that silences one of the two X chromosomes in the somatic cells of females. We recently used RNA fluorescent in situ hybridization (FISH) to show, in individual nuclei, that marsupial X inactivation (in the absence of XIST) occurs on a gene-by-gene basis, and that escape from inactivation is stochastic and independent of gene location. In the absence of similar data from fibroblast cell lines of eutherian representatives, a meaningful comparison is lacking. We therefore used RNA-FISH to examine XCI in fibroblast cell lines obtained from three distantly related eutherian model species: African savannah elephant (Loxodonta africana), mouse (Mus musculus) and human (Homo sapiens). We show that, unlike the orthologous marsupial X, inactivation of the X conserved region (XCR) in eutherians generally is complete. Two-colour RNA-FISH on female human, mouse and elephant interphase nuclei showed that XCR loci have monoallelic expression in almost all nuclei. However, we found that many loci located in the evolutionarily distinct recently added region (XAR) displayed reproducible locus-specific frequencies of nuclei with either one or two active X alleles. We propose that marsupial XCI retains features of an ancient incomplete silencing mechanism that was augmented by the evolution of the XIST gene that progressively stabilized the eutherian XCR. In contrast, the recently added region of the eutherian X displays an incomplete inactivation profile similar to that observed on the evolutionarily distinct marsupial X and the independently evolved monotreme X chromosomes.

Fig. 1

RNA-FISH of an X-borne gene (red) and an autosomal control (green) in male and female fibroblasts of three eutherian species. Transcription from the two autosomal alleles (green) is detected in all nuclei. a Male fibroblast nuclei show transcription from the single X-borne allele, whereas female fibroblast nuclei show transcription from either one (b) or two (c) X-borne ATRX alleles. Nuclei are counterstained with DAPI (blue). HSA Homo sapiens, LAF Loxodonta africana, MMU Mus musculus

Fig. 2

Activity maps of the human (HSA), elephant (LAF) and mouse (MMU) X chromosomes. Bars represent percentage of nuclei in which the X-borne test loci are transcribing from two (blue), one (red) or zero (grey) alleles. X conserved region is indicate in black. X added region is indicated in white