Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells.

Proper placement of epigenetic marks on DNA and histones is fundamental to normal development, and perturbations contribute to a variety of disease states. Combinations of marks act together to control gene expression; therefore, detecting their colocalization is important, but because of technical challenges, such measurements are rarely reported. Instead, measurements of epigenetic marks are typically performed one at a time in a population of cells, and their colocalization is inferred by association. Here, we describe a single-molecule analytical approach that can perform direct detection of multiple epigenetic marks simultaneously and use it to identify mechanisms coordinating placement of three gene silencing marks, trimethylated histone H3 lysine 9, lysine 27 (H3K9me3, H3K27me3), and cytosine methylation (mC), in the normal and cancer genome. We show that H3K9me3 and mC are present together on individual chromatin fragments in mouse embryonic stem cells and that half of the H3K9me3 marks require mC for their placement. In contrast, mC and H3K27me3 coincidence is rare, and in fact, mC antagonizes H3K27me3 in both embryonic stem cells and primary mouse fibroblasts, indicating this antagonism is shared among primary cells. However, upon immortalization or tumorigenic transformation of mouse fibroblasts, mC is required for complete H3K27me3 placement. Importantly, in human promyelocytic cells, H3K27me3 is also dependent on mC. Because aberrant placement of gene silencing marks at tumor suppressor genes contributes to tumor progression, the improper dependency of H3K27me3 by mC in immortalized cells is likely to be fundamental to cancer. Our platform can enable other studies involving coordination of epigenetic marks and leverage efforts to discover disease biomarkers and epigenome-modifying drugs.

Coordinate regulation of DNA methylation and H3K27me3 in mouse embryonic stem cells.

Chromatin is separated into functional domains distinguished by combinatorial patterns of post-translational histone modifications and DNA methylation. Recent studies examining multiple histone modifications have found numerous chromatin states with distinct profiles of chromatin marks and functional enrichments. There are data showing coordinate regulation between DNAme and H3K27me3, which are both involved in the establishment and maintenance of epigenetic gene silencing, but the data are conflicting. Multiple studies have presented evidence to support the theory that PRC2 and DNAme cooperate to achieve silencing, or alternatively that H3K27me3 and DNAme act antagonistically. Here we examine the effect loss of either PRC2 or DNA methyltransferase activity has on the placement of the reciprocal mark in mouse ES cells. We find that DNAme is acting globally to antagonize the placement of H3K27me3, in accordance with recently published results. At least 471,011 domains in the mouse genome acquire H3K27me3 when DNAme is diminished. Of these 466,563 have been shown to be fully methylated in wildtype ES cells, indicating the effects of DNAme on H3K27me3 are direct. In a reciprocal experiment, we examine the effect loss of PRC2 has on the placement of DNAme. In contrast to the global antagonism DNAme has on the placement of H3K27me3, loss of H3K27me3 has a modest effect on DNAme, with only 4% of genes undergoing changes in DNAme, including 861 showing increases and 552 showing losses of overall DNAme. We anticipate that integrating genomic datasets where the effect of loss of a particular epigenetic mark has on the placement of other marks will help elucidate the rules governing epigenetic regulation and what role coordinate regulation of epigenetic marks plays in development and disease.

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.

Successful computational prediction of novel imprinted genes from epigenomic features.

Approximately 100 mouse genes undergo genomic imprinting, whereby one of the two parental alleles is epigenetically silenced. Imprinted genes influence processes including development, X chromosome inactivation, obesity, schizophrenia, and diabetes, motivating the identification of all imprinted loci. Local sequence features have been used to predict candidate imprinted genes, but rigorous testing using reciprocal crosses validated only three, one of which resided in previously identified imprinting clusters. Here we show that specific epigenetic features in mouse cells correlate with imprinting status in mice, and we identify hundreds of additional genes predicted to be imprinted in the mouse. We used a multitiered approach to validate imprinted expression, including use of a custom single nucleotide polymorphism array and traditional molecular methods. Of 65 candidates subjected to molecular assays for allele-specific expression, we found 10 novel imprinted genes that were maternally expressed in the placenta.

An essential gene mutagenesis screen across the highly conserved piebald deletion region of mouse chromosome 14.

The piebald deletion complex is a set of overlapping chromosomal deficiencies on distal mouse chromosome 14. We surveyed the functional genetic content of the piebald deletion region in an essential gene mutagenesis screen of 952 genomes to recover seven lethal mutants. The ENU-induced mutations were mapped to define genetic intervals using the piebald deletion panel. Lethal mutations included loci required for establishment of the left-right embryonic axis and a loss-of-function allele of Phr1 resulting in respiratory distress at birth. A functional map of the piebald region integrates experimental genetic data from the deletion panel, mutagenesis screen, and the targeted disruption of specific genes. A comparison of several genomic intervals targeted in regional mutagenesis screens suggests that the piebald region is characterized by a low gene density and high essential gene density with a distinct genomic content and organization that supports complex regulatory interactions and promotes evolutionary stability.