The consequences of global hypomethylation at repetitive elements in cancer has long been the subject of speculation regarding the generation of genomic instability and potential activation of oncogenes. Ever since studies on viable yellow agouti (Avy
) mice revealed that hypomethylation of a retrotransposon could induce ectopic expression of a gene and influence disease susceptibility 
it has been postulated that similar events may occur in humans. While hypomethylation during tumorigenesis occurs quite frequently, a direct demonstration of the impact of hypomethylation of repetitive elements on gene expression has not been conducted. Transcriptome sequencing has recently revealed the prevalence of transcripts originating from alternate TSS within repetitive elements in humans, indicating a potential functional role of activated repeats in altering gene expression 
. Active L1s were mostly found in embryonic and cancerous tissues, many of which result alternate transcripts of protein-coding genes. Using several specific L1s we have demonstrated the mechanism of transcriptional activation and, taken together with the results of Faulkner et al
, our results highlight the previously underappreciated impact of hypomethylation on ectopic gene expression, possibly contributing to tumorigenesis in a synergistic or cooperative manner (see model in ).
Model of the epigenetic alterations that occur between inactive L1s and active L1s during tumorigenesis.
To elucidate the mechanism of transcriptional activation of repetitive elements, we compared the epigenetic alterations, including methylation status, histone modifications, and nucleosome positioning, that occur at a single copy of an L1 between a transcriptionally inactive and active state. Since current methods did not exist for such a study we employed several novel assays, including using primers able to amplify specific L1s, enabling methylation and ChIP assays to be performed on single copies, and a modification of the method for determining nucleosome positioning at a single molecule resolution, currently limited to unmethylated CpG islands, which allowed for the determination of nucleosome positioning in a methylated region. We were able to show that transcription from the L1 promoter is silenced by DNA methylation, providing direct evidence that one function of DNA methylation is to protect the human genome from retrotransposons.
Transcriptional activation of L1 promoters by hypomethylation results in a chromatin structure similar to that of active single copy genes such as p16, revealing that the features of active promoters, such as acquisition of active histone marks, H2A.Z, and nucleosome free regions upstream of TSSs, are not restricted to canonical gene promoters. In addition, we found that the unique structure of the L1 promoter results in two very stable nucleosome occupancy states, the inactive tetranucleosome structure and the active dinucleosome structure, and that hypomethylation could result in a switch between the two. It has been demonstrated that tetranucleosomes form a compact chromatin fiber 
. Therefore, the widespread chromatin remodeling due to global hypomethylation of L1 promoters could contribute to chromosomal instability through the loss of many stabilizing tetranucleosome structures.
To our knowledge we have provided the first direct evidence that transcriptional activation of repetitive elements is caused by hypomethylation and chromatin remodeling at their promoters, occurs in a human diseased state, and may play a role in disease predisposition. Specifically, hypomethylation of a L1 promoter induces an alternate transcript of the MET
oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. The presence of L1-MET
hypomethylation across the entire urothelium of tumor-bearing bladders has several possible explanations. Epigenetic alterations such as hypermethylation of tumor suppressor genes and hypomethylation of L1s have been found in normal epithelia adjacent to several types of tumors, including breast 
, esophageal 
, and colon 
, indicating the presence of a “field defect”. Our data supports the presence of an epigenetic field defect in bladders with cancer, either due to independent events across the urothelium or clonal expansion 
. However, another possible explanation is that the loss of L1-MET
methylation occurred during early development before the bladder was fully formed. While some evidence for such abnormal epigenetic programming exists, as a recent study revealed that people who develop bladder cancer have slightly lower levels of global DNA methylation in their blood than healthy control cases 
, we did not find any evidence of a loss of methylation at global L1s or specific L1s in our patient WBC samples (Figure S9
). Another possibility, which cannot be ruled out by this data, is that the presence of a tumor causes epigenetic changes across the bladder.
Whatever the underlying mechanism, the modulation of gene expression by hypomethylation of a retrotransposon such as what occurs at the agouti locus in mice is also found in humans. This leads to the activation of surrounding genes, which may contribute to tumorigenesis in a synergistic or cooperative manner. Transurethral resection of bladder tumors would leave behind large areas of epigenetically altered urothelium, possibly contributing to the high level of recurrence of bladder cancer. Fortunately, hypomethylation at specific L1s seems to provide a valuable biomarker that has the potential to significantly impact the diagnosis and treatment of bladder cancer.