Limited evolutionary conservation of imprinting in the human placenta - PubMed (original) (raw)

Limited evolutionary conservation of imprinting in the human placenta

D Monk et al. Proc Natl Acad Sci U S A. 2006.

Abstract

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Schematic representation of the KCNQ1 domain on human chromosome 11p15.5 showing the relative organization of genes and CpG islands. The methylation status of all promoter CpG islands was examined in liver-, muscle-, lymphocyte-, and placenta-derived DNA. Methylation patterns were assessed first by restriction digestion of bisulphite PCR products. The positions of the restriction sites used are shown (↓). Example of patterns obtained by sequencing bisulphite PCR product obtained from placenta-derived DNA. Similar patterns were obtained in all tissues analyzed. Each circle represents a single CpG dinucleotide on the strand, a methylated cytosine (•) or an unmethylated cytosine (○). For clarity, only the first 20 CpG dinucleotides from each CpG island are shown. In all tissues analyzed, the only evidence for differential methylation was at the human KvDMR1/KCNQIOT1 promoter; all other promoter CpG islands were hypomethylated. The imprinted expression for all genes in the cluster was analyzed in first-trimester fetal tissues, term placenta, and cytokeratin 7-positive trophoblast cells. DNA sequence traces for heterozygous term placentae samples are shown for all genes and the resulting RT-PCR sequences. Open boxes depict genes biallelic in all tissues throughout gestation, whereas red boxes represents ubiquitously imprinted, maternally expressed genes. The hatched red box presents the maternal-specific expression of KCNQ1 limited to first-trimester fetal tissues only. The blue hatched box symbolizes the paternal-specific expression of the KCNQ1OT1 transcript observed in first-trimester material only. Both of these genes are biallelically expressed in term placenta.

Fig. 2.

Alelle-specific analysis of histone modifications in term placentae by using antibodies against H3K4me2, H3K9ac, H3K9me2, and H3K27me3. For each ChIP, representative data for the placental (pl) and parental (♂ and ♀) genotypes, ChIP input (In), unbound fraction (U), and antibody-bound (B) are shown. SNP accession no. and locations are given in Table 3, which is published as supporting information on the PNAS web site. The relative enrichment (∗, >2-fold) of the maternal allele in precipitated H3K4me2 (CDKN1C 2.1; SLC22A18 4.1; no enrichment at PHLDA2 exon 2) and H3K9ac (CDKN1C 7.3; SLC22A18 2.1; PHLDA2 2.5), and for the paternal allele in precipitated H3K9me2 (CDKN1C 2.8; SLC22A18 2.3; PHLDA2 4.4) and H3K27me3 (CDKN1C 10.1; SLC22A18 6.9; PHLDA2 3.6), is limited to genes with ubiquitous maternal-specific expression. An opposite allelic pattern was observed at the KvDMR1 with relative enrichment of paternal H3K4me2 (3.9) and H3K9ac (2.9) and maternal H3K9me2 (2.1) and H3K27me3 (5.0) precipitation. No enrichment was observed within the transcribed KCNQ1OT1 region.

Fig. 3.

Schematic map of the IGF2R region on human chromosome 6. The methylation status of the CpG islands within this domain was analyzed by bisulphite sequencing and by Southern blotting (data not shown). The only differentially methylated region found maps to the intronic CpG island within intron 2 of the IGF2R gene. The allele-specific expression was analyzed in first-trimester and term placenta samples. Both IGF2R and SLC22A2 show polymorphic imprinting in term placenta (horizontal hatched red boxes), whereas SLC22A3 exhibits temporal imprinting, with monoallelic expression limited to first-trimester placenta (diagonal hatched red box). As in the mouse, SLC22A1 was biallelically expressed. No DNA methylation differences were observed between imprinted and nonimprinted samples (data not shown, an example of the methylation profile from a placenta sample showing imprinted expression is given).

Fig. 4.

Absence of placental imprinting at the human GATM and DCN genes. (A) The human GATM gene maps to human chromosome 15q21.1 and its mouse orthologue exhibits maternal-specific expression limited to placenta. The human GATM promoter is hypomethylated in all tissues analyzed (bisulphite sequence data from placenta-derived DNA shown), and the gene is biallelically expressed in all fetal and term samples when analyzing coding SNPs in either exon 3 or the 3′ UTR. No allele-specific histone modifications were detected within the promoter region or exon 3. (B) The human DCN gene maps to human chromosome 12q21.33, and the mouse orthologue displays maternal-specific expression limited to the placenta. Using a SNP in the 3′ UTR, all isoforms of DCN were biallelically expressed. The figure shows the DNA sequence trace used for genotyping and a subsequent RT-PCR that amplifies all isoforms. No allele-specific histone modifications were detected within the promoter region.

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Limited evolutionary conservation of imprinting in the human placenta - PubMed (original) (raw)