Epigenetic memory in induced pluripotent stem cells.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Common DNA methylation alterations in multiple brain regions in autism.

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders defined clinically by a triad of features including impairment in social interaction, impairment in communication in social situations and restricted and repetitive patterns of behavior and interests, with considerable phenotypic heterogeneity among individuals. Although heritability estimates for ASD are high, conventional genetic-based efforts to identify genes involved in ASD have yielded only few reproducible candidate genes that account for only a small proportion of ASDs. There is mounting evidence to suggest environmental and epigenetic factors play a stronger role in the etiology of ASD than previously thought. To begin to understand the contribution of epigenetics to ASD, we have examined DNA methylation (DNAm) in a pilot study of postmortem brain tissue from 19 autism cases and 21 unrelated controls, among three brain regions including dorsolateral prefrontal cortex, temporal cortex and cerebellum. We measured over 485,000 CpG loci across a diverse set of functionally relevant genomic regions using the Infinium HumanMethylation450 BeadChip and identified four genome-wide significant differentially methylated regions (DMRs) using a bump hunting approach and a permutation-based multiple testing correction method. We replicated 3/4 DMRs identified in our genome-wide screen in a different set of samples and across different brain regions. The DMRs identified in this study represent suggestive evidence for commonly altered methylation sites in ASD and provide several promising new candidate genes.

Epigenetic stochasticity, nuclear structure and cancer: the implications for medicine.

The aim of this review is to summarize an evolution of thinking about the epigenetic basis of human cancer, from the earliest studies of altered DNA methylation in cancer to the modern comprehensive epigenomic era. Converging data from epigenetic studies of primary cancers and from experimental studies of chromatin in development and epithelial-mesenchymal transition suggest a role for epigenetic stochasticity as a driving force of cancer, with Darwinian selection of tumour cells at the expense of the host. This increased epigenetic stochasticity appears to be mediated by large-scale changes in DNA methylation and chromatin in domains associated with the nuclear lamina. The implications for diagnosis include the potential to identify stochastically disrupted progenitor cells years before cancer develops, and to target drugs to epigenetic drivers of gene expression instability rather than to mean effects per se.

Hot-stop PCR: a simple and general assay for linear quantitation of allele ratios.

We have developed a simple, quantitative assay for measurement of allele ratios that circumvents the problem of heteroduplex formation skewing the results of restriction endonuclease digestion of PCR products. This assay, 'hot-stop PCR', involves addition of a radiolabelled PCR primer at the final cycle. We applied the assay to analysis of loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) in tumours.

Human KVLQT1 gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements.

Genomic imprinting is an epigenetic chromosomal modification in the gamete or zygote causing preferential expression of a specific parental allele in somatic cells of the offspring. We and others have identified three imprinted human genes on 11p15.5, IGF2, H19, and p57KIP2, although the latter gene is separated by 700 kb from the other two, and it is unclear whether there are other imprinted genes within this large interval. We previously mapped an embryonal tumour suppressor gene to this region, as well as five balanced germline chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS), a condition characterized by prenatal overgrowth and cancer. We isolated the upstream exons of the previously identified gene KVLQT1, which causes the familial cardiac defect long-QT (LQT) syndrome. We found that KVLQT1 spans much of the interval between p57KIP2 and IGF2, and that it is also imprinted. We demonstrated that the gene is disrupted by chromosomal rearrangements in BWS patients, as well as by a balanced chromosomal translocation in an embryonal rhabdoid tumour. Furthermore, the lack of parent-of-origin effect in LQT syndrome appears to be due to relative lack of imprinting in the affected tissue, cardiac muscle, representing a novel mechanism for variable penetrance of a human disease gene.

Simple purification of human chromosomes to homogeneity using muntjac hybrid cells.

Chromosome sorting from hybrid cells offers enormous advantages for gene mapping and cloning, but purification of most chromosomes has been largely hindered by their similarity in size to other chromosomes. We have developed a novel cell line and strategy that allows simple, mass purification of mammalian chromosomes, permitting significant target genome enrichment. This strategy takes advantage of the small number of giant chromosomes (1,2,X) of the female Indian muntjac, a barking deer, avoiding the problem of size similarity. We introduced human chromosomes into a cell line derived from a muntjac and purified them to homogeneity using a relatively simple technique. This strategy should facilitate the isolation of chromosomes from species other than human for which hybrid cells are not available currently.

Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms' tumour.

The insulin-like growth factor-II (IGF2) and H19 genes are imprinted in mouse and human, with expression of the paternal IGF2 and maternal H19 alleles. IGF2 undergoes loss of imprinting (LOI) in most Wilms' tumours (WT). We now show that: (i) LOI of IGF2 is associated with a 80-fold down regulation of H19 expression; (ii) these changes are associated with alterations in parental-origin-specific, tissue-independent sites of DNA methylation in the H19 promoter; and (iii) loss of heterozygosity is also associated with loss of H19 expression. Thus, imprinting of a large domain of the maternal chromosome results in a reversal to a paternal epigenotype. These data also suggest an epigenetic mechanism for inactivation of H19 as a tumour suppressor gene.

Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia.

Expression of Hoxa7 and Hoxa9 is activated by proviral integration in BXH2 murine myeloid leukaemias. This result, combined with the mapping of the HOXA locus to human chromosome 7p15, suggested that one of the HOXA genes might be involved in the t(7;11)(p15;p15) translocation found in some human myeloid leukaemia patients. Here we show that in three patients with t(7;11), the chromosome rearrangement creates a genomic fusion between the HOXA9 gene and the nucleoporin gene NUP98 on chromosome 11p15. The translocation produces an invariant chimaeric NUP98/HOXA9 transcript containing the amino terminal half of NUP98 fused in frame to HOXA9. These studies identify HOXA9 as an important human myeloid leukaemia gene and suggest an important role for nucleoporins in human myeloid leukaemia given that a second nucleoporin, NUP214, has also been implicated in human myeloid leukaemia.

Microallelotyping defines the sequence and tempo of allelic losses at tumour suppressor gene loci during colorectal cancer progression.

Microallelotyping of many regions from individual colorectal tumours was used to determine the sequence and tempo of allelic loss on 5q, 17p and 18q during neoplastic progression. No allelic losses were found in normal tissues surrounding colorectal neoplasms, but losses occurred abruptly on 5q at the transition from normal colonic epithelium to the benign adenoma, and on 17p at the transition from adenoma to carcinoma, indicating an essential role for these losses in tumour progression. Allelic losses were uniform throughout extensively microdissected benign adenomas and carcinomas. However, substantial allelic heterogeneity was found in high-grade dysplasia, the transition lesion between adenoma and carcinoma. Thus, allelic losses on 5q and 17p are associated with abrupt waves of clonal neoplastic expansion, and high-grade dysplasia is characterized by a high degree of allelic heterogeneity.

Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability.

Loss of imprinting (LOI) is an epigenetic alteration of some cancers involving loss of parental origin-specific expression of imprinted genes. We observed LOI of the insulin-like growth factor-II gene in twelve of twenty-seven informative colorectal cancer patients (44%), as well as in the matched normal colonic mucosa of the patients with LOI in their cancers, and in peripheral blood samples of four patients. Ten of eleven cancers (91%) with microsatellite instability showed LOI, compared with only two of sixteen tumors (12%) without microsatellite instability (P < 0.001). Control patients without cancer showed LOI in colonic mucosa of only two of sixteen cases (12%, P < 0.001) and two of fifteen blood samples (13%, P < 0.001). These data suggest that LOI in tumor and normal tissue identifies most colorectal cancer patients with microsatellite instability in their tumors, and that LO! may identify an important subset of the population with cancer or at risk of developing cancer.

Detection of haplotype-dependent allele-specific DNA methylation in WGBS data.

In heterozygous genomes, allele-specific measurements can reveal biologically significant differences in DNA methylation between homologous alleles associated with local changes in genetic sequence. Current approaches for detecting such events from whole-genome bisulfite sequencing (WGBS) data perform statistically independent marginal analysis at individual cytosine-phosphate-guanine (CpG) sites, thus ignoring correlations in the methylation state, or carry-out a joint statistical analysis of methylation patterns at four CpG sites producing unreliable statistical evidence. Here, we employ the one-dimensional Ising model of statistical physics and develop a method for detecting allele-specific methylation (ASM) events within segments of DNA containing clusters of linked single-nucleotide polymorphisms (SNPs), called haplotypes. Comparisons with existing approaches using simulated and real WGBS data show that our method provides an improved fit to data, especially when considering large haplotypes. Importantly, the method employs robust hypothesis testing for detecting statistically significant imbalances in mean methylation level and methylation entropy, as well as for identifying haplotypes for which the genetic variant carries significant information about the methylation state. As such, our ASM analysis approach can potentially lead to biological discoveries with important implications for the genetics of complex human diseases.

Nanoelectromechanics of methylated DNA in a synthetic nanopore.

Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a <2 nm diameter pore, which we attribute to the stretching transition; this can differ by >1 V/20 nm depending on the methylation level, but not the DNA sequence.

Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors.

A novel strategy to determine the clonal origin of human tumors has been devised. The strategy involves the use of a cloned polymorphic X-chromosomal gene and two restriction endonucleases. The first endonuclease distinguishes the paternal and maternal copies of the gene through a DNA polymorphism of restriction fragment length. The second endonuclease distinguishes active from inactive copies of this gene through changes in DNA methylation. As illustrations of this strategy, three human cancers were each shown to be monoclonal. The analysis described should have a wide variety of clinical and experimental applications.

Hypomethylation of DNA from benign and malignant human colon neoplasms.

The methylation state of DNA from human colon tissue displaying neoplastic growth was determined by means of restriction endonuclease analysis. When compared to DNA from adjacent normal tissue, DNA from both benign colon polyps and malignant carcinomas was substantially hypomethylated. With the use of probes for growth hormone, gamma-globin, alpha-chorionic gonadotropin, and gamma-crystallin, methylation changes were detected in all 23 neoplastic growths examined. Benign polyps were hypomethylated to a degree similar to that in malignant tissue. These results indicate that hypomethylation is a consistent biochemical characteristic of human colonic tumors and is an alteration in the DNA that precedes malignancy.

Mutation affecting the 12th amino acid of the c-Ha-ras oncogene product occurs infrequently in human cancer.

A point mutation alters the 12th amino acid of the c-Ha-ras oncogene product p21 in a human bladder cancer cell line. This is, at present, the only mutation known to result in a human transforming gene. This mutation may therefore represent a possible target for mutagenesis leading to carcinogenesis in humans. By means of restriction enzyme analysis, 29 human cancers, including 20 primary tumor tissues, derived from organs commonly exposed to environmental carcinogens, were tested for the presence of this mutation. None of ten primary bladder carcinomas exhibited the mutation; nor did nine colon carcinomas or ten carcinomas of the lung. Thus the point mutation affecting the 12th amino acid of the c-Ha-ras gene product, while a valuable model for carcinogenesis, does not appear to play a role in the development of most human epithelial cancers of the bladder, colon, or lung.

Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11.

A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cell. As a test of the method, several overlapping STFs from 11p15 were shown to cause in vitro growth arrest of rhabdomyosarcoma cells. This activity mapped between the beta-globin and insulin genes.

Concerted nonsyntenic allelic loss in human colorectal carcinoma.

Familial polyposis coli (FPC) is caused by an autosomal dominant gene on chromosome 5, and it has been proposed that colorectal cancer in the general population arises from loss or inactivation of the FPC gene, analogous to recessive tumor genes in retinoblastoma and Wilms' tumor. Since allelic loss can be erroneously scored in nonhomogeneous samples, tumor cell populations were first microdissected from 24 colorectal carcinomas, an additional nine cancers were engrafted in nude mice, and nuclei were flow-sorted from an additional two. Of 31 cancers informative for chromosome 5 markers, only 6 (19%) showed loss of heterozygosity of chromosome 5 alleles, compared to 19 of 34 (56%) on chromosome 17, and 17 of 33 (52%) on chromosome 18. Therefore, it appears that (i) FPC is a true dominant for adenomatosis but not a common recessive gene for colon cancer; and (ii) simple Mendelian models involving loss of alleles at a single locus may be inappropriate for understanding common human solid tumors.

Tissue, developmental, and tumor-specific expression of divergent transcripts in Wilms tumor.

The Wilms tumor locus on chromosome 11p13 has been mapped to a region defined by overlapping, tumor-specific deletions. Complementary DNA clones representing transcripts of 2.5 (WIT-1) and 3.5 kb (WIT-2) mapping to this region were isolated from a kidney complementary DNA library. Expression of WIT-1 and WIT-2 was restricted to kidney and spleen. RNase protection revealed divergent transcription of WIT-1 and WIT-2, originating from a DNA region of less than 600 bp. Both transcripts were present at high concentrations in fetal kidney and at much reduced amounts in 5-year-old and adult kidneys. Eleven of 12 Wilms tumors classified as histopathologically heterogeneous exhibited absent or reduced expression of WIT-2, whereas only 4 of 14 histopathologically homogeneous tumors showed reduced expression. These data demonstrate a molecular basis for the pathogenetic heterogeneity in Wilms tumorigenesis.