Distinct Patterns of Gene-Specific Methylation in Mammalian Placentas: Implications for Placental Evolution and Function

Location of methylation assays used in the current study. Alignment of methylation assays was performed using the BLAT function of the UCSC genome browser (human genome build GRCh37). Chromosomal location is listed along with the relative positions and level of sequence homology between the different methylation assays. The 5′ end of genes of interest is shown along with the transcription start site (arrow). Red dashes denote individual CpG sites within each assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Methylation of the APC gene in 7 eutherian placentas. Following bisulphite conversion of placental genomic DNA, regions of interest were amplified with primer sequences specific to converted DNA sequence. Amplified PCR products were then cloned into a DNA vector before transformation into E. coli. Individual colonies were then randomly selected for DNA isolation and sequencing. Biological replicate samples from independent placentas (number listed in parentheses) were tested for each species. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. Grey horizontal bars denote CpG island regions associated with the gene of interest and transcription start site (raised arrows). Black bars show locations of associated exonic regions. Red dashes denote individual CpG sites within each assay which correspond to circles below. Circled methylation data are presented for a single placental sample (as an example) for which 8–12 DNA sequences from individual alleles (horizontal rows) were generated. Due to space constraints, only 8 alleles from each placental sample are shown. Shaded circles denote methylated CpG sites within specific DNA clones (open circles). Mean methylation (0–100%) for each CpG site in biological replicates (number in brackets) is shown as shaded bars below each site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Methylation of the SFRP2 gene in 7 eutherian placentas. Bisulphite sequencing was carried out as described in Fig. 2. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. For details see Fig. 2.

Methylation of the CYP24A1 gene in 7 eutherian placentas. Bisulphite sequencing was carried out as described in Fig. 2. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. For details see Fig. 2.

Methylation of the DNMT1 gene in 7 eutherian placentas. Bisulphite sequencing was carried out as described in Fig. 2. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. For details see Fig. 2.

Placenta specific gene methylation levels according to phylogenetic relationship (A) Phylogenetic relationship of eutherians showing placenta barrier type (He, Hemochorial; Hm, Hemomonochorial; Hd, Hemodichorial; Ht, Hemotrichorial; Ep, Epitheliochorial; En, Endotheliochorial), placenta shape (C; Cotyledonary; Z, Zonary; D, Diffuse; Dd, Discoid; Bd, Bidiscoid), and type of interdigitation (T, Trabecular; Lb, Labyrinthine; F, Folded; L, Lamellar). Species examined in this study are highlighted in red. (B) Summary of methylation level for each of the four genes examined in this study for seven different species. Phylogenetic tree adapted from {Wildman, 2006 #1672}. Note that the classification of manatee placental barrier type has recently been modified from He to En {Carter, 2008 #1673}. Classification of methylation profile is based on parameters listed in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).