Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer

Abstract

It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.

Figure 1. Methylation and expression of L1-MET correlates in cell lines.

(A) Map of alternate transcript from L1-MET. Exons are represented by black boxes and a red box represents the specific L1. The bent arrows indicate transcriptional start sites and ATGs indicate translational start sites. Horizontal arrows indicate the primers for PCR of bisulfite converted DNA and RT–PCR. The bisulfite-specific primers Bi-L1-5′ and Bi-MET-3′ were used to amplify L1-MET for methylation analysis and Bi-L1-5′ and Bi-L1-3′ for global L1 methylation analysis. The RT–PCR primers, RT-L1-MET-5′ and RT-MET-3′ were used to amplify cDNA of the L1-MET transcript for expression analysis and RT-MET-3′ and RT-MET-5′ for the full length MET expression analysis. The lower tick marks represent each CpG site. Vertical arrows indicate the CpG sites analyzed by the Ms-SNuPE assay. (B) L1-MET methylation (red bars) and L1 methylation (black bars) was analyzed by Ms-SNuPE in 8 normal tissues, one normal bladder fibroblast cell line (LD419), two non-tumorigenic urothelial cell lines (UROtsa and NK2426), and 20 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1-MET from technical triplicates. Error bars represent the standard deviation. (C) Expression of L1-MET was measured using real-time RT PCR in one normal bladder fibroblast cell line, two normal urothelial cell lines and 10 bladder carcinoma cell lines. There is clearly a strong correlation between DNA methylation and expression in all 13 cell lines examined. Values are the average from technical duplicates. Red bars indicate the methylation status of L1-MET, which is also represented in (B), and green bars represent the level of expression relative to GAPDH.

Figure 2. DNA methylation silences the L1-MET promoter.

(A) Map of the CpG sites (represented by the lower tick marks) within the L1-MET anti-sense promoter (ch7:116364010–116364564), which was ligated into a CpG-less luciferase vector (pCpGL) in both orientations, allowing for the measurement of either L1-MET activity (red bars) or L1 activity (black bars). (B) The relative luciferase activity (firefly luciferase light units/Renilla luciverase light units) is represented as the mean ± SD and was high in the untreated vector, the methyl donor S-adenosyl-methionine (SAM) alone, and the CpG methyltransferase (SssI) alone. When the methyltransferase enzyme and the methyl donor (SssI+SAM) were added to the luciferase vectors together then promoter activity was silenced in both directions. The values are the average of three biological replicates. Error bars represent the standard deviation.

Figure 3. Chromatin remodeling occurs at an active L1-MET promoter.

(A) DNA methylation at L1-MET and global L1s was determined by pyrosequencing in the immortalized urothelial cell line UROtsa and bladder carcinoma cell line T24. Chromatin immunoprecipitation was performed using antibodies for H3K4me3, acetylated H3, and H2A.Z. The values of the ChIP assay are the average of three experiments with technical duplicates. Error bars represent the standard deviation, and p16 represents a single copy gene control. The presence of active histone marks was associated with absence of DNA methylation at L1-MET in the cancer cell line. Methylase dependent single promoter analysis (MSPA) with M. CviPI, a GpC methyltransferase, of the (B) endogenously methylated L1-MET promoter (ch7:116364020–116364664) in the UROtsa immortalized urothelial cell line and the (C) endogenously unmethylated L1-MET promoter in T24 bladder carcinoma cells. (D) DNA methylation at L1-MET and global L1s was determined by pyrosequencing in the colon cancer cell line HCT116 and HCT116 DKO cells (DNMT1 hypomorph/DNMT3B knockout) , . Chromatin immunoprecipitation was performed using antibodies for H2A.Z. The presence of active histone marks was associated with absence of DNA methylation at L1-MET in the DKO cell line. Methylase dependent single promoter analysis (MSPA) with M. CviPI, a GpC methyltransferase, of the (E) endogenously methylated L1-MET promoter in HCT116 colon cancer cells, and (F) endogenously unmethylated L1-MET promoter in HCT116 DKO cells. White circles indicate unmethylated sites and black circles indicate methylated sites. Orange bars indicate areas of protection consistent with the presence of a nucleosome.

Figure 4. Nucleosome eviction is a frequent occurrence at L1 promoters.

Partial MNase digestion of nucleosomes was followed by fractionation by a sucrose density gradient. When a Southern for genomic DNA was performed on the DNA in each fraction (6–16), enrichment in the mono- and dinucleosome fractions was revealed. When a Southern for L1s was performed enrichment of L1s in the di- and tetranucleosome fractions was found. According to our model the L1 promoters with a tetranucleosomal structure should be inactive and methylated.

Figure 5. Methylation and expression status of L1-MET correlates in bladder tissues.

Horizontal lines represent the mean and n the number of patient samples. (A) Methylation status was analyzed by Ms-SNuPE in normal tissues (N, green), corresponding normal tissues (CN, dark blue), and bladder tumors (T, red). Values are an average of two CpG sites. (B) Expression of the alternate transcript from L1-MET and (C) the host gene MET, and the control gene GAPDH was measured by real-time RT–PCR. *** represents p<0.001, ** represents p<0.01, and * represents p<0.05 as determined by the Mann-Whitney test. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA, the results show a consistent trend.

Figure 6. Methylation of L1-MET across the bladder.

(A) Tissue samples were taken from five patients of their tumors (red, T) and at increasing distances from the tumor (0.5 to 2 cm) in the surrounding normal-appearing tissue in multiple directions (light blue, a to d). Additionally, distant normal-appearing samples were taken at least 5 cm from the tumor (dark blue, C). (B) Methylation at L1-MET and (C) global L1 was measured by pyrosequencing. The green line represents the mean methylation of normal samples from cancer-free patients. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA, the results show a consistent trend. (D) Bisulfite sequencing of L1-MET was performed on samples from two bladder cancer-free patients (#4987 and #5240) and one bladder cancer patient (#6519). White circles represent unmethylated CpGs and black circles represent methylated CpGs.

Figure 7. Model of the epigenetic alterations that occur between inactive L1s and active L1s during tumorigenesis.

An L1 promoter is usually silenced by DNA methylation and has a compact chromatin structure with four nucleosomes occupying the promoter. Upon hypomethylation during tumorigenesis the L1 promoter becomes transcriptionally active. The active promoter loses a nucleosome upstream of each of the transcription start sites, resulting in a dinucleosome structure. The remaining nucleosomes have acetylated H3, H3K4me3, and H2A.Z. (−1) represents the nucleosome directly upstream of the transcription start site, while (+1) represents directly downstream nucleosome of transcriptional start site.