Elsevier

Gene

Volume 448, Issue 2, 15 December 2009, Pages 151-167
Gene

Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements

https://doi.org/10.1016/j.gene.2009.08.006Get rights and content

Abstract

Close to 50% of the human genome harbors repetitive sequences originally derived from mobile DNA elements, and in normal cells, this sequence compartment is tightly regulated by epigenetic silencing mechanisms involving chromatin-mediated repression. In cancer cells, repetitive DNA elements suffer abnormal demethylation, with potential loss of silencing. We used a genome-wide microarray approach to measure DNA methylation changes in cancers of the head and neck and to compare these changes to alterations found in adjacent non-tumor tissues. We observed specific alterations at thousands of small clusters of CpG dinucleotides associated with DNA repeats. Among the 257,599 repetitive elements probed, 5% to 8% showed disease-related DNA methylation alterations. In dysplasia, a large number of local events of loss of methylation appear in apparently stochastic fashion. Loss of DNA methylation is most pronounced for certain members of the SVA, HERV, LINE-1P, AluY, and MaLR families. The methylation levels of retrotransposons are discretely stratified, with younger elements being highly methylated in healthy tissues, while in tumors, these young elements suffer the most dramatic loss of methylation. Wilcoxon test statistics reveals that a subset of primate LINE-1 elements is demethylated preferentially in tumors, as compared to non-tumoral adjacent tissue. Sequence analysis of these strongly demethylated elements reveals genomic loci harboring full length, as opposed to truncated elements, while possible enrichment for functional LINE-1 ORFs is weaker. Our analysis suggests that, in non-tumor adjacent tissues, there is generalized and highly variable disruption of epigenetic control across the repetitive DNA compartment, while in tumor cells, a specific subset of LINE-1 retrotransposons that arose during primate evolution suffers the most dramatic DNA methylation alterations.

Introduction

The DNA of most tumors has a reduced content of methylated cytosine residues. This so-called global “hypomethylation” affects primarily DNA sequences that belong to interspersed DNA repeats. In normal human tissues, DNA repeats are predominantly methylated, consistent with the requirement to maintain genomic stability by transcriptional silencing of retroelements whose potential deleterious functions include DNA mobilization as well as the facilitation of recombination events in somatic cells. There have been a considerable number of reports of transcriptional activation of retrotransposons in the context of loss of DNA methylation. Expression of human endogenous retroviruses (HERVs) has been detected in breast cancer (Wang-Johanning et al., 2001), ovarian cancer (Menendez et al., 2004, Wang-Johanning et al., 2007), leukemia cell lines (Patzke et al., 2002), and urothelial and renal cell carcinomas (Florl et al., 1999). Increased transcriptional expression of HERV-K has been reported in teratocarcinoma (Löwer et al., 1984, Herbst et al., 1998), breast cancer cells and adjacent tissues (Wang-Johanning et al., 2003, Golan et al., 2008), and in melanoma (Muster et al., 2003, Büscher et al., 2006, Serafino et al., 2009). Stauffer et al. (2004) used massively parallel signature sequencing (MPSS) to define the number and type of transcripts of endogenous retroviruses of the LTR family in various cancers. This study reported that HERV-H, a relatively young retrotransposon, was expressed in cancers of the intestine, bone marrow, bladder, and cervix and was more highly expressed than the other families in cancers of the stomach, colon, and prostate. Recently Alves et al. (2008) have reported that a specific HERV-H element present in the X chromosome is selectively transcribed in 60% of colon cancers and in a high proportion of metastatic colon cancers. There is evidence for context-specific induction of LINE-1 transcription during oxidative stress (Teneng et al., 2007). In a relatively large study of squamous head and neck carcinomas, Smith et al. (2007) reported that the DNA methylation level of LINE-1 elements was significantly reduced and correlated with environmental insults such as alcohol use and smoking, as well as tumor stage.

Here we report a systematic study of DNA methylation changes occurring in the repetitive DNA compartment of squamous carcinomas of the head and neck. In contrast to previous studies, we use a novel microarray-based approach to obtain discrete DNA methylation data at hundreds of thousands of individual repetitive DNA loci in the human genome. We then use extensive annotation resources for different subfamilies of repeats to evaluate possible relationships between loss of epigenetic silencing in the context of natural history of cancer and the evolutionary history of repetitive element sub-compartments in the human genome.

Section snippets

Materials and methods

A microarray analysis method developed in our laboratory permits genome-wide assessment of DNA methylation status using restriction endonucleases (see Supplementary Materials Section 1). Among the 339,314 probes in the microarray, 257,599 are dedicated to the measurement of the methylation levels of individual members of interspersed DNA repeat families.

Methylation patterns of major classes and families of DNA repeats

We analyzed the DNA methylation profiles of 33 tumors and 17 non-tumor adjacent tissue samples obtained from patients with head and neck squamous carcinoma (HNSCC). We also generated DNA methylation profiles from the buccal epithelia of 10 normal individuals, which served as controls. In addition, an analysis of sperm DNA was performed in technical triplicate to assess the reproducibility of the microarray results. We calculated an average methylation value for selected subsets of “genomic

Conclusions

A novel microarray method for analysis of DNA methylation, based on the use of methylation-sensitive as well as methylation-dependent endonucleases, enables the interrogation of methylation levels in all compartments of the genome, including repetitive elements. Analysis of a substantial set of samples of squamous carcinomas of the head and neck, as well as non-tumor adjacent tissue and normal controls, reveals a complex framework of epigenetic dysregulation, where loss of methylation

Acknowledgments

We wish to thank Vincent Marchesi for his interest and support of this work. This work was supported by a pilot grant from the Yale Cancer Center and the Swebilius Foundation, as well as by NIH grant no. R21 CA116079-01, awarded to P.M.L. Furthermore, S.S. and J.R. were supported by an NIH training grant NLM T15 LM07056.

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