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

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.

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

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.

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 primate-restricted 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.

In silico tandem affinity purification refines an Oct4 interaction list.

INTRODUCTION: Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency. METHODS: In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels. RESULTS: Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors. CONCLUSIONS: Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.