The drivers of gene expression maintain a fragile balance that tends to cross the LINE in cancer. LINE-1s are transposable elements full of both mystery and regulatory potential. Within their 5’ untranslated region (UTR) lies a bidirectional promoter (L1-5’UTR) that consists of a sense promoter (L1-SP), which transcribes the element, and an antisense promoter (L1-ASP), which drives transcription in the other direction. Thus, a transposition event can drive the expression of novel transcripts; however, these elements are rarely examined at an individual level.
Previously, the lab of Cristina Tufarelli at the University of Nottingham (UK) discovered LINE-1 chimeric transcripts (LCTs), driven the by L1-ASP, which consist of both LINE-1 and a unique sequence taken along for the ride. The team focused on two LCTs arising from intergenic regions (LCT-13 and LCT-14) to demonstrate a functional role in cancer, where they discovered that LCT-13 silences a tumor suppressor. Now, the team set their sights on whether a loss of methylation in the L1-5’UTR of LCT-13 and LCT-14 activated their expression in colorectal cancer (CRC). To accomplish this, the team examined matched tumor and normal tissue from six CRC patients, CRC cell lines, and breast cancer cell lines as another comparison point.
Here’s what went down:
- Bisulfite sequencing and qRT-PCR revealed that LCT-13 and LCT-14 methylation and expression aren’t inversely correlated in most CRC patients, as they can be methylated and
- The team then probed deeper with a few sanity checks:
- 5’ rapid amplification of cDNA ends (RACE) of LCT-13 confirmed that the L1-ASP is the promoter for LCT-13 and that DNA methylation (5mC) alone doesn’t prevent transcription.
- Since standard bisulfite sequencing can’t discriminate 5mC from its oxidized form (5hmC), the team employed hydroxymethylated DNA immunoprecipitation (hMeDIP) to show that there is no increase in 5hmC at LCT-13 and LCT-14.
- HCT116 cells treated with the DNA methylation inhibitor 5-azacytidine (5-aza) show no change in LCT-13 expression and neither do cells with a double knockout for DNMT1 and DNMT3B; however, cells with a knockout for just one of those methyltransferases displayed decreased LCT-13 expression.
- Given the observed complexities in the relationship between methylation and expression, the team set their sights on histone modifications and found that H4K20me3 exhibited the expected inverse association with expression at LCT-13 and LCT-14.
- H4K20me3 was also a better indicator of 5-aza sensitivity.
Overall, the findings demonstrate that LINE-1’s can be both methylated and active and that H4K20me3 can play an even more important role than DNA methylation in the regulation of LCTs. Together, these findings demonstrate the novel insight to be gained by studying LINE-1 promoters at an individual level.
Go read between the LINEs in Epigenetics, March 2017.