Random X inactivation and extensive mosaicism in human placenta revealed by analysis of allele-specific gene expression along the X chromosome

Abstract

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Figure 1. Summary of allele-specific X-linked gene expression in human placenta.

Ratios of expressed alleles for each locus are shown: red (0∶100); pink (between 0∶100 and 20∶80); green (above 20∶80). Ratios of expressed alleles were scored by PeakPicker or visual analysis (*) of electropherograms. GM135, completed skewed XCI human fibroblast cell line. Placental samples (pl.) are grouped as showing predominantly completely skewed, skewed, or random inactivation. (ND) not determined. (-) non-informative locus; (X) informative locus; (M) expression from maternal allele; (P) expression from paternal allele. Column one: gene symbol; Column two: chromosomal position as in Vega Human View, v35 - Mar 2009 (http://vega.sanger.ac.uk/Homo_sapiens/index.html). Column three: SNP variant according to NCBI dbSNP BUILD129 (http://www.ncbi.nlm.nih.gov/SNP/). Column four: Gene expression on the Xi, where expression results are indicated as the number of primary human fibroblasts expressing each gene from the Xi per number fibroblasts tested, or (§) number of rodent/human somatic cell hybrids with the Xi that expressed the gene per number of hybrids tested .

Figure 2. Analyses of expressed alleles in human term placenta.

Examples of electropherograms of DNA and cDNA sequences of X-linked SNPs in (A) completely skewed fibroblast GM135; (B) pl.19; (C) pl.28; and (D) pl.17. Genes symbols and corresponding SNP ID are indicated above. Sequences from DNA and cDNA from cell line/placental samples, and corresponding maternal (mat.) DNA are shown. SNP position is highlighted in yellow. In (A) ZFX is shown as an example of a gene that escapes XCI.

Figure 3. Quantification of ratio of expressed alleles using PeakPicker software.

Solid lines indicate threshold levels for 0∶100 (lower) and 50∶50 (upper) ratios of expressed alleles. Dotted line indicates theoretical ratio of 20∶80. Open circles represent data from genomic DNA, filled circles from cDNA (filled triangles are experimental replicas), and asterisks from cDNA of completely skewed fibroblast GM135. Gene symbols and corresponding SNP ID are indicated. Analysis of (A) ZFX (escapes XCI), (B) TCEAL4 and (C) GPC4 (subjected to XCI) genes in different placentas; (D) Ratios of expressed alleles of different genes in pl.05 – threshold levels and theoretical ratio of 20∶80 is shown for each gene.

Figure 4. Mosaicism of the human full-term placenta regarding XCI.

Electropherograms of DNA and cDNA sequences of X-linked SNPs in three different fragments of placentas (A) 31, (B) 32 and (C) 33. Gene symbols and corresponding SNP ID are indicated. SNPs are highlighted in yellow.