Genomic map of candidate human imprint control regions: the imprintome

Abstract

Imprinted genes - critical for growth, metabolism, and neuronal function - are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.

Keywords: Epigenetics; foetal origins; genomic imprinting; imprint control regions; methylation; whole genome.

Figure 1.

Detection of ICRs genome-wide. A bioinformatics selection algorithm, putICR, was used to identify methylation fractions using whole-genome bisulphite sequencing (WGBS) of DNA from kidney, liver, and brain embryonic tissues and gametes (i.e., sperm and oocyte). (a) Genome coverage was >20X for the somatic tissues in more than 75% of the fraction base sampled. The genome coverage was lower in the available oocyte gametic sequence data (accession number JGAS00000000006), but higher in sperm because our data were supplemented with control sperm data from PRJNA754049 [25,26]. (b) The size range of candidate imprint control regions (ICRs) averaged 248 bp. (c) This is similar to previously identified ICRs (Avg: 375 bp) [14].

Figure 2.

Previously known and candidate ICRs. (a) PLAGL1 (ICR_404) and (b) MEG3 (ICR_872,873) imprinted genes contain previously known ICRs (blue boxes) [14]. They were also identified in our whole-genome bisulphite sequencing (WGBS) screen for candidate ICRs (red boxes); visualize at https://humanicr.org/. The ICRs defined herein overlap and extend beyond the currently known ICRs. (c) PTCHD3 (ICR_643) and (d) MCTS2P/HM13 (ICR_1191) contain only candidate ICRs (red boxes) determined in this study, and potentially control novel imprinted genes. The methylation levels for ICRs is 50 ± 15% for the candidate ICRs. DNA methylation levels determined by WGBS in sperm and oocytes are also provided. These sequence data were supplemented with publicly available gametic sequence (accession number JGAS00000000006) and part of the control sperm data from PRJNA754049 [25,26]. Dots indicate hemi-methylation (green), hypomethylation (blue), and hypermethylation (yellow) based on WGBS. The vertical dashed red lines delineate the candidate ICR regions.

Figure 3.

Pyrosequencing results for previously known ICRs. DNA methylation profiles determined by whole genome bisulphite sequencing (WGBS) are shown for imprinted genes (a) PEG3/ZIM2 (ICR_1142) and (b) PEG10 (ICR_475) with previously known (blue boxes) and candidate ICRs (red boxes); visualize at https://humanicr.org/. DNA methylation levels determined by WGBS in sperm and oocytes are also provided. These sequence data were supplemented with publicly available gametic sequence (accession number JGAS00000000006) and part of control sperm data from PRJNA754049 [25,26]. Dots indicate hemi-methylation (green), hypomethylation (blue) and hypermethylation (yellow) based on (WGBS). The vertical dashed red lines delineate the candidate ICR regions. Confirmation of 50 ± 15% DNA methylation of the ICRs determined by WGBS is shown for (c) PEG3/ZIM2 and (d) PEG10 using pyrosequencing of kidney (black circles), liver (red circles), and brain (blue circles) tissues from 24 embryos. Confirmation of 50 ± 15% DNA methylation determined by WGBS is shown for (e) PEG3/ZIM2 and (f) PEG10 using pyrosequencing for CD14 monocytes (red squares) and HUVECs (black squares) from 14 adult individuals.

Figure 4.

Candidate ICR for DHRSX (ICR_1394) in the pseudo autosomal region, PAR1, on the X chromosome; visualize at https://humanicr.org/. Whole genome bisulphite sequencing (WGBS) identified a candidate ICR (red box) in embryonic brain, kidney, and liver tissue for a candidate imprinted gene associated with starvation induced autophagy [45]. DNA methylation determined by WGBS in sperm and oocytes is also shown; these sequence data were supplemented with publicly available gametic sequence (accession number JGAS00000000006) and part of control sperm data from PRJNA754049 [25,26]. Dots indicate hemi-methylation (green), hypomethylation (blue) and hypermethylation (yellow) based on WGBS. The vertical dashed red lines delineate the candidate ICR regions.