Clara Yujing Cheong

The effect of genotype and in utero environment on interindividual variation in neonate DNA methylomes

Integrating the genotype with epigenetic marks holds the promise of better understanding the biology that underlies the complex interactions of inherited and environmental components that define the developmental origins of a range of disorders. The quality of the in utero environment significantly influences health over the lifecourse. Epigenetics, and in particular DNA methylation marks, have been postulated as a mechanism for the enduring effects of the prenatal environment. Accordingly, neonate methylomes contain molecular memory of the individual in utero experience. However, interindividual variation in methylation can also be a consequence of DNA sequence polymorphisms that result in methylation quantitative trait loci (methQTLs) and, potentially, the interaction between fixed genetic variation and environmental influences. We surveyed the genotypes and DNA methylomes of 237 neonates and found 1423 punctuate regions of the methylome that were highly variable across individuals, termed variably methylated regions (VMRs), against a backdrop of homogeneity. MethQTLs were readily detected in neonatal methylomes, and genotype alone best explained ∼25% of the VMRs. We found that the best explanation for 75% of VMRs was the interaction of genotype with different in utero environments, including maternal smoking, maternal depression, maternal BMI, infant birth weight, gestational age, and birth order. Our study sheds new light on the complex relationship between biological inheritance as represented by genotype and individual prenatal experience and suggests the importance of considering both fixed genetic variation and environmental factors in interpreting epigenetic variation.

Comparison of Methyl-capture Sequencing vs. Infinium 450K methylation array for methylome analysis in clinical samples

ABSTRACT Interindividual variability in the epigenome has gained tremendous attention for its potential in pathophysiological investigation, disease diagnosis, and evaluation of clinical intervention. DNA methylation is the most studied epigenetic mark in epigenome-wide association studies (EWAS) as it can be detected from limited starting material. Infinium 450K methylation array is the most popular platform for high-throughput profiling of this mark in clinical samples, as it is cost-effective and requires small amounts of DNA. However, this method suffers from low genome coverage and errors introduced by probe cross-hybridization. Whole-genome bisulfite sequencing can overcome these limitations but elevates the costs tremendously. Methyl-Capture Sequencing (MC Seq) is an attractive intermediate solution to increase the methylome coverage in large sample sets. Here we first demonstrate that MC Seq can be employed using DNA amounts comparable to the amounts used for Infinium 450K. Second, to provide guidance when choosing between the 2 platforms for EWAS, we evaluate and compare MC Seq and Infinium 450K in terms of coverage, technical variation, and concordance of methylation calls in clinical samples. Last, since the focus in EWAS is to study interindividual variation, we demonstrate the utility of MC Seq in studying interindividual variation in subjects from different ethnicities.

Germline and somatic imprinting in the nonhuman primate highlights species differences in oocyte methylation

Genomic imprinting is an epigenetic mechanism resulting in parental allele-specific gene expression. Defects in normal imprinting are found in cancer, assisted reproductive technologies, and several human syndromes. In mouse models, germline-derived DNA methylation is shown to regulate imprinting. Though imprinting is largely conserved between mammals, species- and tissue-specific domains of imprinted expression exist. Using the cynomolgus macaque (Macaca fascicularis) to assess primate-specific imprinting, we present a comprehensive view of tissue-specific imprinted expression and DNA methylation at established imprinted gene clusters. For example, like mouse and unlike human, macaque IGF2R is consistently imprinted, and the PLAGL1, INPP5F transcript variant 2, and PEG3 imprinting control regions are not methylated in the macaque germline but acquire this post-fertilization. Methylome data from human early embryos appear to support this finding. These suggest fundamental differences in imprinting control mechanisms between primate species and rodents at some imprinted domains, with implications for our understanding of the epigenetic programming process in humans and its influence on disease.

Transcriptome changes affecting Hedgehog and cytokine signalling in the umbilical cord: implications for disease risk.

Background Babies born at lower gestational ages or smaller birthweights have a greater risk of poorer health in later life. Both the causes of these sub-optimal birth outcomes and the mechanism by which the effects are transmitted over decades are the subject of extensive study. We investigated whether a transcriptomic signature of either birthweight or gestational age could be detected in umbilical cord RNA. Methods The gene expression patterns of 32 umbilical cords from Singaporean babies of Chinese ethnicity across a range of birthweights (1698–4151 g) and gestational ages (35–41 weeks) were determined. We confirmed the differential expression pattern by gestational age for 12 genes in a series of 127 umbilical cords of Chinese, Malay and Indian ethnicity. Results We found that the transcriptome is substantially influenced by gestational age; but less so by birthweight. We show that some of the expression changes dependent on gestational age are enriched in signal transduction pathways, such as Hedgehog and in genes with roles in cytokine signalling and angiogenesis. We show that some of the gene expression changes we report are reflected in the epigenome. Conclusions We studied the umbilical cord which is peripheral to disease susceptible tissues. The results suggest that soma-wide transcriptome changes, preserved at the epigenetic level, may be a mechanism whereby birth outcomes are linked to the risk of adult metabolic and arthritic disease and suggest that greater attention be given to the association between premature birth and later disease risk.

Alternative Splicing in Self-Renewal of Embryonic Stem Cells

Much of embryonic stem cell biology has focused on transcriptional expression and regulation of genes that could mediate its unique potential in self-renewal or pluripotency. In alignment with our present understanding on the genetic, protein, and epigenetic factors that may direct cell fate, we present a short overview of the often overlooked contribution of alternative splice variants to regulatory diversity. Progressing beyond the limitations of a fixed genomic sequence, alternative splicing offers an additional layer of complexity to produce protein variants that may differ in function and localization that can direct embryonic stem cells to specific differentiation pathways. In light of the number of variants that can be produced at key ES cell genes alone, it is challenging to consider how much more multifaceted transcriptional regulation truly is, and if this can be captured more fully in future works.

Infinium Monkeys: Infinium 450K Array for the Cynomolgus Macaque (Macaca fascicularis)

The Infinium Human Methylation450 BeadChip Array (Infinium 450K) is a robust and cost-efficient survey of genome-wide DNA methylation patterns. Macaca fascicularis (Cynomolgus macaque) is an important disease model; however, its genome sequence is only recently published, and few tools exist to interrogate the molecular state of Cynomolgus macaque tissues. Although the Infinium 450K is a hybridization array designed to the human genome, the relative conservation between the macaque and human genomes makes its use in macaques feasible. Here, we used the Infinium 450K array to assay DNA methylation in 11 macaque muscle biopsies. We showed that probe hybridization efficiency was related to the degree of sequence identity between the human probes and the macaque genome sequence. Approximately 61% of the Human Infinium 450K probes could be reliably mapped to the Cynomolgus macaque genome and contain a CpG site of interest. We also compared the Infinium 450K data to reduced representation bisulfite sequencing data generated on the same samples and found a high level of concordance between the two independent methodologies, which can be further improved by filtering for probe sequence identity and mismatch location. We conclude that the Infinium 450K array can be used to measure the DNA methylome of Cynomolgus macaque tissues using the provided filters. We also provide a pipeline for validation of the array in other species using a simple BLAST-based sequence identify filter.

Alterations to DNA methylation and expression of CXCL14 are associated with suboptimal birth outcomes

CXCL14 is a chemokine that has previously been implicated in insulin resistance in mice. In humans, the role of CXCL14 in metabolic processes is not well established, and we sought to determine whether CXCL14 is a risk susceptibility gene important in fetal programming of metabolic disease. For this purpose, we investigated whether CXCL14 is differentially regulated in human umbilical cords of infants with varying birth weights. We found an elevated expression of CXCL14 in human low birth weight (LBW) cords, as well as in cords from nutritionally restricted Macaca fascicularis macaques. To further analyze the regulatory mechanisms underlying the expression of CXCL14, we examined CXCL14 in umbilical cord-derived mesenchymal stem cells (MSCs) that provide an in vitro cell-based system amenable to experimental manipulation. Using both whole frozen cords and MSCs, we determined that site-specific CpG methylation in the CXCL14 promoter is associated with altered expression, and that changes in methylation are evident in LBW infant-derived umbilical cords that may indicate future metabolic compromise through CXCL14.

Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primaterestricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/ SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of “simple” cancer-associated chromosome deletions.

Cell type-specific DNA methylation in neonatal cord tissue and cord blood: a 850K-reference panel and comparison of cell types

ABSTRACT Accounting for cellular heterogeneity is essential in neonatal epigenome-wide association studies (EWAS) performed on heterogeneous tissues, such as umbilical cord tissue (CT) or cord blood (CB). Using a reference-panel-based statistical approach, the cell type composition of heterogeneous tissues can be estimated by comparison of whole tissue DNA methylation profiles with cell type-specific DNA methylation signatures. Currently, there is no adequate DNA methylation reference panel for CT, and existing CB panels have been generated on lower coverage Infinium HumanMethylation450 arrays. In this study, we generate a reference panel for CT and improve available CB panels by using the higher coverage Infinium MethylationEPIC arrays. We performed DNA methylation profiling of 9 cell types isolated from CT and CB samples from 14 neonates. In addition to these cell types, we profiled DNA methylation of unfractionated CT and CB. Cell type composition of these unfractionated tissue samples, as estimated by our reference panels, was in agreement with that obtained by flow cytometry. Expectedly, DNA methylation profiles from CT and CB were distinct, reflecting their mesenchymal and hematopoietic stem cell origins. Variable CpGs from both unfractionated CT and its isolated cell types were more likely to be located in open seas and intronic regions than those in CB. Cell type specific CpGs in CT were enriched in intercellular matrix pathways, while those from CB were enriched in immune-related pathways. This study provides an open source reference panel for estimation and adjustment of cellular heterogeneity in CT and CB, and broadens the scope of tissue utilization assessed in future neonatal EWAS studies.