L1 retrotransposition is activated by Ten-eleven-translocation protein 1 and repressed by methyl-CpG binding proteins

Abstract

One of the major functions of DNA methylation is the repression of transposable elements, such as the long-interspersed nuclear element 1 (L1). The underlying mechanism(s), however, are unclear. Here, we addressed how retrotransposon activation and mobilization are regulated by methyl-cytosine modifying ten-eleven-translocation (Tet) proteins and how this is modulated by methyl-CpG binding domain (MBD) proteins. We show that Tet1 activates both, endogenous and engineered L1 retrotransposons. Furthermore, we found that Mecp2 and Mbd2 repress Tet1-mediated activation of L1 by preventing 5hmC formation at the L1 promoter. Finally, we demonstrate that the methyl-CpG binding domain, as well as the adjacent non-sequence specific DNA binding domain of Mecp2 are each sufficient to mediate repression of Tet1-induced L1 mobilization. Our study reveals a mechanism how L1 elements get activated in the absence of Mecp2 and suggests that Tet1 may contribute to Mecp2/Mbd2-deficiency phenotypes, such as the Rett syndrome. We propose that the balance between methylation "reader" and "eraser/writer" controls L1 retrotransposition.

Keywords: 5-hydroxymethylcytosine; DNA methylation; Rett syndrome; genome stability; repetitive elements.

Figure 1.

Tet1 reactivates retrotransposition of endogenous L1. (A) Schematic overview of L1 retrotransposition. (B) Experimental rationale of human L1 retrotransposition detection. (C) Relative 5hmC levels in L1 5′UTR (n = 3, *p < 0.05, independent 2-sample student's t-test), (D) relative L1 transcription levels (n ≥ 4; ns = non significant; ***p < 0.001, independent 2-sample student's t-test) and (E) relative L1 ORF2 DNA content was checked 48 hours after Tet1CD, Tet1CDmut and mock transfection (n = 4, *p < 0.05, independent 2-sample student's t-test). (F) Scheme summarizing the effect of Tet1 on the 3 steps of L1 retrotransposition. Black and red circles indicate 5mC and 5hmC nucleotides, respectively. Bars represent the mean + standard deviation (SD). (G) Experimental rationale of mouse L1 retrotransposition detection. (H) Boxplot of the log2-fold changes of the triple Tet-knockout mouse embryonic stem cells relative to wild-type (V6.5) for all genes and all L1 elements. Negative values indicate a downregulation in the knockout relative to the wild-type, positive values an upregulation. Significant elements are marked in color. The red line is at zero, i.e., the expected value if expression were identical in the wild type and mutant. (I) Relative mouse L1 ORF2 content. Bar represent mean+ SD. (n = 3, *p < 0.05, independent 2-sample student's t-test).

Figure 2.

Tet1 reactivates retrotransposition of engineered L1. (A) Experimental rationale. The ratio of EGFP-positive cells in Tet1/mcherry-positive and negative cells, was quantified to detect recent L1 retrotransposition events. (B-C) Relative increases of retrotransposition events (B) 24 hours and (C) 48 hours after Tet1CD, Tet1CDmut and mcherry transfection. At least 3 independent experiments were performed and more than 250,000 cells for each group were analyzed and the bar represents the mean + SD. Independent 2-sample student's t-test was performed between Tet1CD and Tet1CDmut or Tet1CD and mcherry transfected cells. Only significant differences were indicated on the plots as 2 asterisks (p < 0.005). (D) Representative images of the pLRE3-EGFP reporter cell line 48 hours after mcherry-Tet1CD/CDmut and mcherry transfection, respectively. Scale bar: 100 µm.

Figure 3.

MBD proteins prevent Tet-mediated reactivation of endogenous L1. (A) Relative 5hmC in L1 5′UTR (n = 3, *p < 0.05, independent 2-sample student's t-test), (B) relative L1 transcription levels (n = 4; **p < 0.01, independent 2-sample student's t-test) and (C) relative L1 ORF2 DNA content were checked 48 hours after cotransfection with plasmids coding for Tet1CD- and MBD proteins (n = 3, *p < 0.05, independent 2-sample student's t-test). All of the 3 independent experiments for IDTRD and Mbd2 showed decreased, but variable L1 copy number when compared with Tet1CD, giving rise to an apparent non-significant difference. (D) Scheme illustrating the effect of Tet1- and MBD proteins on L1 retrotransposition. Black and red circles indicate 5mC and 5hmC nucleotides, respectively. Bars represent the mean + SD.