Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription

Abstract

In order to explore the defense mechanism by which retrotransposons are repressed, we assessed the ability of methyl-CpG-binding protein 2, MeCP2, to influence LINE-1 (L1) and Alu transcription and, furthermore, L1 retrotransposition. In transient transfection assays, targeting of the transcriptional-repression domain (TRD) of MeCP2 (via a linked Gal4 DNA-binding domain) to the transcriptional start site of L1 promoter-driven reporter constructs efficiently repressed transcription. The Gal4-linked TRD of the related methyl-CpG-binding protein MBD1 also repressed transcription but not that of MBD2. Furthermore, full-length MeCP2 effectively repressed transcription of a HpaII-methylated L1 reporter. Secondly, we used a genetic assay employing a full-length neo-marked L1 reporter construct to study L1 retrotransposition. We found the Gal4-linked TRD of MeCP2 to repress effectively L1 retrotransposition when targeted to the retrotransposition reporter. Retrotransposition was also reduced in response to in vitro HpaII methylation of the reporter and was further decreased by co-expressed full-length MeCP2. In striking contrast expression of the Gal4-linked TRD of MeCP2 had no inhibiting effect on transcription of an AluSx reporter tagged with a 7S-upstream sequence. Furthermore, full-length MeCP2 abrogated the methylation-induced repression of this reporter. Our results indicate that MeCP2 serves a role in repression of L1 expression and retrotransposition but has no inhibiting effect on Alu transcription.

Figure 1

The TRDs of MeCP2 and MBD1 repress L1 transcription. (A) Schematic maps of the reporters L1.3/L1_RP-Luc (L1-Luc) and G5L1.3/L1_RP-Luc (G5L1-Luc) and the expression constructs Gal4-TRD_MeCP2, Gal4-TRD_MBD1 and Gal4-TRD_MBD2. Numbers indicate relevant amino acids of the MBD protein portion in each fusion, and the respective TRDs are shown as hatched boxes. (B) Immunoblot analysis of HEK293 cells transfected with expression constructs Gal4-TRD_MeCP2, Gal4-TRD_MBD1 or Gal4-TRD_MBD2 (0.1 µg each) using an anti-Gal4BD monoclonal antibody. The fusion proteins exhibit apparent molecular masses of 57, 46 and 39 kDa, respectively. (C) HEK293 cells were transfected with reporter constructs L1.3-Luc or L1_RP-Luc that did or did not contain upstream Gal4 DNA-binding sites (G5) and with or without the indicated amounts (0.06–0.4 µg) of expression construct Gal4-TRD_MeCP2. Relative luciferase activity of reporter constructs lacking Gal4 DNA-binding sites co-transfected with 0.1 µg of plasmid Gal4-TRD_MeCP2 was set as 1.0. Columns represent mean luciferase activities (± standard deviations) of three to five independent experiments. (D) HEK293 cells were co-transfected with reporter plasmids L1.3-Luc or G5L1.3-Luc and expression construct Gal4-TRD_MeCP2 (0.1 µg). After 24 h, transfected cells were treated with TSA (100 ng/ml) and incubated for another 24 h. (E) HEK293 cells were co-transfected with reporter constructs L1.3-Luc or G5L1.3-Luc and expression constructs Gal4-TRD_MBD1 or Gal4-TRD_MBD2 (0.1 µg each).

Figure 2

MeCP2 represses transcription from a methylated L1 promoter. (A) Schematic representation of the sites (nucleotides 36, 101, 304 and 481) in the L1.3–5′UTR methylated by M.HpaII methylase. (B) HEK293 cells were co-transfected with unmethylated (U) or HpaII-methylated (Me) reporter L1.3-Luc and expression constructs encoding full-length FLAG-tagged MeCP2, MBD1v3 or MBD2b. Relative luciferase activity of the unmethylated reporter in the absence of coexpressed genes was set as 1.0. Columns represent mean luciferase activities (± standard deviations) of three to five independent experiments. (C) Expression of FLAG-tagged MeCP2, MBD1v3 and MBD2b was controlled for by immunoblot analysis with an anti-FLAG monoclonal antibody. The tagged proteins exhibit apparent molecular masses of 81, 81 and 29 kDa, respectively.

Figure 3

The TRD of MeCP2 represses L1 retrotransposition. (A) Reporter construct JM101/L1.2ΔCMV or G5JM101/L1.2ΔCMV was co-transfected with the empty vector pcDNA1.1 or with expression construct Gal4-TRD_MeCP2 into HeLa cells. Selection with G418 began 3 days after transfection and, after 12 days, G418 resistant foci were fixed and stained with Giemsa for visualization. Results of a representative transposition assay are shown. (B) Effect of Gal4-TRD_MeCP2 on relative transposition frequencies of the neo-marked L1.2 reporters. The bar chart represents the data in Table 1A. Relative retrotransposition frequencies refer to the mean retrotransposition rate of construct JM101/L1.2ΔCMV (357 ± 17 × 10⁶), which is set as 1.0. (C) Expression of Gal4-TRD_MeCP2 72 h post-transfection was controlled for by immunoblot analysis with anti-Gal4BD antibody. The upper 57-kDa band represents the full-length Gal4-TRD_MeCP2. The lower band very likely results from cellular protease activity, since the C-terminal half of MeCP2 exhibits prominent sensitivity to proteolysis (10).

Figure 4

Methylated and overexpressed full-length MeCP2 diminishes L1 retrotransposition frequency. (A) Unmethylated (U) or HpaII-methylated (Me) reporter JM101/L1.2ΔCMV was co-transfected with empty vector pcDNA1.1 or with the expression construct for FLAG-tagged MeCP2. Results of a representative transposition assay are shown. (B) Effect of MeCP2 on relative retrotransposition frequencies of the methylated versus unmethylated neo-marked L1.2 reporter. The bar chart is based on the data in Table 1B. Relative retrotransposition frequencies refer to the mean retrotransposition rate of the unmethylated reporter JM101/L1.2ΔCMV (612 ± 40 × 10⁶), which is set as 1.0. (C) Expression of FLAG-tagged MeCP2 (81 kDa) 72 h post-transfection was controlled for by immunoblot analysis with anti-FLAG antibody.

Figure 5

Effects of MeCP2 on Alu transcription. (A) Schematic diagram of the reporters ^7SLAluSx^TMBC1 and G5-^7SLAluSx^TMBC1. The location of promoter elements box A and box B, the adenine-rich region and the diagnostic unique BC1 region are indicated. (B) Effects of different TRDs on ^7SLAluSx transcription. HEK293 cells were transfected with reporter construct ^7SLAluSx^TMBC1 or G5-^7SLAluSx^TMBC1 and expression constructs Gal4-TRD_MeCP2, Gal4-TRD_MBD1 or Gal4-TRD_MBD2 or no expression construct. (C) Effects of methylation and full-length MeCP2 on ^7SLAluSx transcription. HEK293 cells were transfected with unmethylated or SssI methylated ^7SLAluSx^TMBC1 and with or without an expression construct encoding full-length FLAG-tagged MeCP2. An autoradiogram of a northern dot blot analyzing three independent transfections is shown. In (B), expression levels using the G5 reporter are expressed as the change (% ± standard deviation) relative to the expression levels using the reporter without Gal4 DNA-binding sites.