Hengmi Cui

The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores

Alterations in DNA methylation (DNAm) in cancer have been known for 25 years, including hypomethylation of oncogenes and hypermethylation of tumor suppressor genes1. However, most studies of cancer methylation have assumed that functionally important DNAm will occur in promoters, and that most DNAm changes in cancer occur in CpG islands2,3. Here we show that most methylation alterations in colon cancer occur not in promoters, and also not in CpG islands but in sequences up to 2 kb distant which we term “CpG island shores.” CpG island shore methylation was strongly related to gene expression, and it was highly conserved in mouse, discriminating tissue types regardless of species of origin. There was a surprising overlap (45-65%) of the location of colon cancer-related methylation changes with those that distinguished normal tissues, with hypermethylation enriched closer to the associated CpG islands, and hypomethylation enriched further from the associated CpG island and resembling non-colon normal tissues. Thus, methylation changes in cancer are at sites that vary normally in tissue differentiation, and they are consistent with the epigenetic progenitor model of cancer4, that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer.

DNMT1 and DNMT3b cooperate to silence genes in human cancer cells

Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.

Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA

Tumour suppressor genes (TSGs) inhibiting normal cellular growth are frequently silenced epigenetically in cancer. DNA methylation is commonly associated with TSG silencing, yet mutations in the DNA methylation initiation and recognition machinery in carcinogenesis are unknown. An intriguing possible mechanism for gene regulation involves widespread non-coding RNAs such as microRNA, Piwi-interacting RNA and antisense RNAs. Widespread sense–antisense transcripts have been systematically identified in mammalian cells, and global transcriptome analysis shows that up to 70% of transcripts have antisense partners and that perturbation of antisense RNA can alter the expression of the sense gene. For example, it has been shown that an antisense transcript not naturally occurring but induced by genetic mutation leads to gene silencing and DNA methylation, causing thalassaemia in a patient. Here we show that many TSGs have nearby antisense RNAs, and we focus on the role of one RNA in silencing p15, a cyclin-dependent kinase inhibitor implicated in leukaemia. We found an inverse relation between p15 antisense (p15AS) and p15 sense expression in leukaemia. A p15AS expression construct induced p15 silencing in cis and in trans through heterochromatin formation but not DNA methylation; the silencing persisted after p15AS was turned off, although methylation and heterochromatin inhibitors reversed this process. The p15AS-induced silencing was Dicer-independent. Expression of exogenous p15AS in mouse embryonic stem cells caused p15 silencing and increased growth, through heterochromatin formation, as well as DNA methylation after differentiation of the embryonic stem cells. Thus, natural antisense RNA may be a trigger for heterochromatin formation and DNA methylation in TSG silencing in tumorigenesis.

Intra-individual change over time in DNA methylation with familial clustering.

CONTEXT Changes over time in epigenetic marks, which are modifications of DNA such as by DNA methylation, may help explain the late onset of common human diseases. However, changes in methylation or other epigenetic marks over time in a given individual have not yet been investigated. OBJECTIVES To determine whether there are longitudinal changes in global DNA methylation in individuals and to evaluate whether methylation maintenance demonstrates familial clustering. DESIGN, SETTING, AND PARTICIPANTS We measured global DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA sampled at 2 visits on average 11 years apart in 111 individuals from an Icelandic cohort (1991 and 2002-2005) and on average 16 years apart in 126 individuals from a Utah sample (1982-1985 and 1997-2005). MAIN OUTCOME MEASURE Global methylation changes over time. RESULTS Twenty-nine percent of Icelandic individuals showed greater than 10% methylation change over time (P < .001). The family-based Utah sample also showed intra-individual changes over time, and further demonstrated familial clustering of methylation change (P = .003). The family showing the greatest global methylation loss also demonstrated the greatest loss of gene-specific methylation by a separate methylation assay. CONCLUSION These data indicate that methylation changes over time and suggest that methylation maintenance may be under genetic control.

BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.

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 LOI may identify an important subset of the population with cancer or at risk of developing cancer.

DNA methylation and genomic imprinting: Insights from cancer into epigenetic mechanisms

Since the discovery of epigenetic alterations in cancer 20 years ago by Feinberg and Vogelstein, a variety of such alterations have been found, including global hypomethylation, gene hypomethylation and hypermethylation, and loss of imprinting (LOI). LOI may precede the development of cancer and may thus serve as a common marker for risk, but also as a model for understanding the developmental mechanism for normal imprinting.

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.

Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages

Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.

BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression

ABSTRACT Chromatin status is characterized in part by covalent posttranslational modifications of histones that regulate chromatin dynamics and direct gene expression. BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein that is thought to play a role in chromatin organization and gene expression. BORIS is a cancer-germ line gene; these are genes normally present in male germ cells (testis) that are also expressed in cancer cell lines as well as primary tumors. This work identifies SET1A, an H3K4 methyltransferase, and BAT3, a cochaperone recruiter, as binding partners for BORIS, and these proteins bind to the upstream promoter regions of two well-characterized procarcinogenic genes, Myc and BRCA1. RNA interference (RNAi) knockdown of BAT3, as well as SET1A, decreased Myc and BRCA1 gene expression but did not affect the binding properties of BORIS, but RNAi knockdown of BORIS prevented the assembly of BAT3 and SET1A at the Myc and BRCA1 promoters. Finally, chromatin analysis suggested that BORIS and BAT3 exert their effects on gene expression by recruiting proteins such as SET1A that are linked to changes in H3K4 dimethylation. Thus, we propose that BORIS acts as a platform upon which BAT3 and SET1A assemble and exert effects upon chromatin structure and gene expression.