Transdifferentiation occurs without resetting development-specific DNA methylation, a key determinant of full-function cell identity.

A number of studies have demonstrated that it is possible to directly convert one cell type to another by factor-mediated transdifferentiation, but in the vast majority of cases, the resulting reprogrammed cells are unable to maintain their new cell identity for prolonged culture times and have a phenotype only partially similar to their endogenous counterparts. To better understand this phenomenon, we developed an analytical approach for better characterizing trans-differentiation-associated changes in DNA methylation, a major determinant of long-term cell identity. By examining various models of transdifferentiation both in vitro and in vivo, our studies indicate that despite convincing expression changes, transdifferentiated cells seem unable to alter their original developmentally mandated methylation patterns.

Pluripotency-independent induction of human trophoblast stem cells from fibroblasts.

Human trophoblast stem cells (hTSCs) can be derived from embryonic stem cells (hESCs) or be induced from somatic cells by OCT4, SOX2, KLF4 and MYC (OSKM). Here we explore whether the hTSC state can be induced independently of pluripotency, and what are the mechanisms underlying its acquisition. We identify GATA3, OCT4, KLF4 and MYC (GOKM) as a combination of factors that can generate functional hiTSCs from fibroblasts.

Muscle injury causes long-term changes in stem-cell DNA methylation.

Injury to muscle brings about the activation of stem cells, which then generate new myocytes to replace damaged tissue. We demonstrate that this activation is accompanied by a dramatic change in the stem-cell methylation pattern that prepares them epigenetically for terminal myocyte differentiation. These de- and de novo methylation events occur at regulatory elements associated with genes involved in myogenesis and are necessary for activation and regeneration.

Chromosomal coordination and differential structure of asynchronous replicating regions.

Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity.

Role of transcription complexes in the formation of the basal methylation pattern in early development.

Following erasure in the blastocyst, the entire genome undergoes de novo methylation at the time of implantation, with CpG islands being protected from this process. This bimodal pattern is then preserved throughout development and the lifetime of the organism. Using mouse embryonic stem cells as a model system, we demonstrate that the binding of an RNA polymerase complex on DNA before de novo methylation is predictive of it being protected from this modification, and tethering experiments demonstrate that the presence of this complex is, in fact, sufficient to prevent methylation at these sites.

Principles of DNA methylation and their implications for biology and medicine.

DNA methylation represents an annotation system for marking the genetic text, thus providing instruction as to how and when to read the information and control transcription. Unlike sequence information, which is inherited, methylation patterns are established in a programmed process that continues throughout development, thus setting up stable gene expression profiles. This DNA methylation paradigm is a key player in medicine.

Postnatal DNA demethylation and its role in tissue maturation.

Development in mammals is accompanied by specific de novo and demethylation events that are thought to stabilize differentiated cell phenotypes. We demonstrate that a large percentage of the tissue-specific methylation pattern is generated postnatally. Demethylation in the liver is observed in thousands of enhancer-like sequences associated with genes that undergo activation during the first few weeks of life.

Programming asynchronous replication in stem cells.

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele.

Epigenetic mechanism of FMR1 inactivation in Fragile X syndrome.

Fragile X syndrome is the most frequent cause of inherited intellectual disability. The primary molecular defect in this disease is the expansion of a CGG repeat in the 5' region of the fragile X mental retardation1 (FMR1) gene, leading to de novo methylation of the promoter and inactivation of this otherwise normal gene, but little is known about how these epigenetic changes occur during development. In order to gain insight into the nature of this process, we have used cell fusion technology to recapitulate the events that occur during early embryogenesis.

Annotating the genome by DNA methylation.

DNA methylation plays a prominent role in setting up and stabilizing the molecular design of gene regulation and by understanding this process one gains profound insight into the underlying biology of mammals. In this article, we trace the discoveries that provided the foundations of this field, starting with the mapping of methyl groups in the genome and the experiments that helped clarify how methylation patterns are maintained through cell division. We then address the basic relationship between methyl groups and gene repression, as well as the molecular rules involved in controlling this process during development in vivo.

Clonally stable Vκ allelic choice instructs Igκ repertoire.

Although much has been done to understand how rearrangement of the Igκ locus is regulated during B-cell development, little is known about the way the variable (V) segments themselves are selected. Here we show, using B6/Cast hybrid pre-B-cell clones, that a limited number of V segments on each allele is stochastically activated as characterized by the appearance of non-coding RNA and histone modifications. The activation states are clonally distinct, stable across cell division and developmentally important in directing the Ig repertoire upon differentiation.

Contribution of epigenetic mechanisms to variation in cancer risk among tissues.

Recently, it was suggested that tissue variation in cancer risk originates from differences in the number of stem-cell divisions underlying each tissue, leading to different mutation loads. We show that this variation is also correlated with the degree of aberrant CpG island DNA methylation in normal cells. Methylation accumulates during aging in a subset of molecules, suggesting that the epigenetic landscape within a founder-cell population may contribute to tumor formation.

DNA Methylation in Cancer and Aging.

DNA methylation is known to be abnormal in all forms of cancer, but it is not really understood how this occurs and what is its role in tumorigenesis. In this review, we take a wide view of this problem by analyzing the strategies involved in setting up normal DNA methylation patterns and understanding how this stable epigenetic mark works to prevent gene activation during development. Aberrant DNA methylation in cancer can be generated either prior to or following cell transformation through mutations.

Maintenance of Epigenetic Information.

The genome is subject to a diverse array of epigenetic modifications from DNA methylation to histone posttranslational changes. Many of these marks are somatically stable through cell division. This article focuses on our knowledge of the mechanisms governing the inheritance of epigenetic marks, particularly, repressive ones, when the DNA and chromatin template are duplicated in S phase.

Tissue-specific DNA demethylation is required for proper B-cell differentiation and function.

There is ample evidence that somatic cell differentiation during development is accompanied by extensive DNA demethylation of specific sites that vary between cell types. Although the mechanism of this process has not yet been elucidated, it is likely to involve the conversion of 5mC to 5hmC by Tet enzymes. We show that a Tet2/Tet3 conditional knockout at early stages of B-cell development largely prevents lineage-specific programmed demethylation events.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome.

Gender-specific postnatal demethylation and establishment of epigenetic memory.

DNA methylation patterns are set up in a relatively fixed programmed manner during normal embryonic development and are then stably maintained. Using genome-wide analysis, we discovered a postnatal pathway involving gender-specific demethylation that occurs exclusively in the male liver. This demodification is programmed to take place at tissue-specific enhancer sequences, and our data show that the methylation state at these loci is associated with and appears to play a role in the transcriptional regulation of nearby genes.

Chronic liver inflammation modifies DNA methylation at the precancerous stage of murine hepatocarcinogenesis.

Chronic liver inflammation precedes the majority of hepatocellular carcinomas (HCC). Here, we explore the connection between chronic inflammation and DNA methylation in the liver at the late precancerous stages of HCC development in Mdr2(-/-) (Mdr2/Abcb4-knockout) mice, a model of inflammation-mediated HCC. Using methylated DNA immunoprecipitation followed by hybridization with "CpG islands" (CGIs) microarrays, we found specific CGIs in 76 genes which were hypermethylated in the Mdr2(-/-) liver compared to age-matched healthy controls.

A novel pax5-binding regulatory element in the igκ locus.

The Igκ locus undergoes a variety of different molecular processes during B cell development, including V(D)J rearrangement and somatic hypermutations (SHM), which are influenced by cis regulatory regions (RRs) within the locus. The Igκ locus includes three characterized RRs termed the intronic (iEκ), 3'Eκ, and Ed enhancers. We had previously noted that a region of DNA upstream of the iEκ and matrix attachment region (MAR) was necessary for demethylation of the locus in cell culture.

Aberrant DNA methylation in ES cells.

Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles.