The human placenta methylome

Abstract

Tissue-specific DNA methylation is found at promoters, enhancers, and CpG islands but also over larger genomic regions. In most human tissues, the vast majority of the genome is highly methylated (>70%). Recently, sequencing of bisulfite-treated DNA (MethylC-seq) has revealed large partially methylated domains (PMDs) in some human cell lines. PMDs cover up to 40% of the genome and are associated with gene repression and inactive chromatin marks. However, to date, only cultured cells and cancers have shown evidence for PMDs. Here, we performed MethylC-seq in full-term human placenta and demonstrate it is the first known normal tissue showing clear evidence of PMDs. We found that PMDs cover 37% of the placental genome, are stable throughout gestation and between individuals, and can be observed with lower sensitivity in Illumina 450K Infinium data. RNA-seq analysis confirmed that genes in PMDs are repressed in placenta. Using a hidden Markov model to map placental PMDs genome-wide and compare them to PMDs in other cell lines, we found that genes within placental PMDs have tissue-specific functions. For regulatory regions, methylation levels in promoter CpG islands are actually higher for genes within placental PMDs, despite the lower overall methylation of surrounding regions. Similar to PMDs, polycomb-regulated regions are hypomethylated but smaller and distinct from PMDs, with some being hypermethylated in placenta compared with other tissues. These results suggest that PMDs are a developmentally dynamic feature of the methylome that are relevant for understanding both normal development and cancer and may be of use as epigenetic biomarkers.

Fig. 1.

Placenta has PMDs covering tissue-specific genes. (A) Methylation levels across the long arm of chromosome 21 in five noncultured human tissues and the H9 hESC line. Smoothing was done using a kernel smoother. (B) The distribution of average percent methylation across 20-kb windows tiling all autosomes. (C) Violin plots showing distribution of methylation levels from four different individuals in HMD and PMD regions from all autosomes on the Infinium 450K array. (D) Representative example on 21q of hidden Markov model-derived PMDs maps (top black bars) based on MethylC-seq data (black landscape plot). In comparison are Infinium 450K data from 21 different third-trimester placental samples (blue landscape plots) and the average of 5 first-trimester (yellow), 6 second-trimester (red), and 21 third-trimester (brown) placental samples. Promoters, CpG islands, and CpG island shores were removed from Infinium data before analysis. (E) Placental PMDs (upper left circle) were analyzed for overlap with previously described PMDs in neuronal cells (SH-SY5Y; upper right circle) and fetal lung fibroblast cells (IMR90; lower circle). These overlaps were used to define seven tissue-specific PMD subtypes: placenta-specific PMDs (P-PMD; red), neuronal-specific PMDs (N-PMD; yellow), lung-specific PMDs (L-PMD; blue), lung-specific HMDs (L-HMD; orange), neuronal-specific HMDs (N-HMD; purple), placenta-specific HMDs (P-HMD; green), and PMDs in all three tissues (all-PMD; brown). The number of genes in each domain type is shown. Tissues with higher expression of genes in L-HMDs, N-HMDs, and P-HMDs by DAVID analysis are diagrammed with arrows. These subdomains are predicted to have genes with functions specific to that tissue type. Benjamini significance values are shown. (F) GO biological process classifications for genes in each domain subtype using DAVID, color-coded as in Fig. 1E. “Sensory perception of “smell,” “neurological system process,” and “cell surface receptor signal transduction” had Benjamini P values of 1.2 × 10⁻²⁶⁵, 4.0 × 10⁻¹⁴⁸, and 1.5 × 10⁻¹⁰⁴, respectively.

Fig. 2.

Genes in PMDs have lower transcript levels. (A) Differences in gene expression between genes in PMDs and HMDs in placenta. Transcript levels are shown as log (FPKM + 1). Only RNA-seq reads that mapped to known transcripts were used for FPKM analysis. (B) Average gene body methylation versus gene expression in placenta. Promoters and CpG islands were removed before computing the average gene body methylation. The histograms to the right show the expression distributions for genes with greater than (red) and less than (black) 70% average gene body methylation. (C) Genomic view of placental data on chromosome 7 showing low levels of transcription within PMDs. The RNA library was not poly-A–selected and reads were mapped to the entire genome using Bowtie.

Fig. 3.

Regulatory elements within PMDs have distinct levels of methylation. DNA methylation analysis for gene regulatory elements within placental PMDs and HMDs. (A–D) Levels of placental DNA methylation within gene bodies (A), promoters (B), promoter CpG islands (C), and gene body CpG islands (D). All four showed significant differences between PMDs and HMDs (Wilcoxon rank sum test P values < 8 × 10⁻¹⁰). For gene body methylation analysis, CpG islands and promoters were first removed. (E) DNA methylation levels on the shores of promoter CpG islands.

Fig. 4.

Polycomb-regulated regions have unique methylation patterns in PMDs and HMDs. (A and B) DNA methylation within the polycomb-regulated DLX5/6 (A) and MSX1 (B) loci for six different tissues. The RNA-seq track is from the non-poly-A selected placental library with reads mapped to the entire genome using Bowtie. RING1B and EZH2 ChIP-seq data tracks are from Ku et al. (25). The hidden Markov model analysis showed entirely within a placental HMD (A) and entirely within a placental PMD (B). (C) Comparison of placenta methylation levels in nonpolycomb HMDs (NH), polycomb HMDs (PH), nonpolycomb PMDs (NP), and polycomb PMDs (PP). For each group, CpG islands were removed before assessing average percent methylation. For nonpolycomb regions, genomic DNA was divided into ≤100-kb subregions for better comparison with polycomb regions. (D) Comparison of non-CpG island methylation levels within polycomb regions between six different tissues. Placenta (red) is distinct from other tissues in methylation of polycomb-regulated regions. (E) Distribution of the lengths of polycomb-regulated regions and PMDs.