CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K(HML-2)

Abstract

A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) transcription remains poorly understood. We investigated in detail the role of CpG methylation on the transcriptional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-K(HML-2) proviral 5' LTRs are methylated at different levels in the cell line Tera-1. Methylation levels correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5' LTR reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5' LTR activity, as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in evolutionary old HERV-K(HML-2) 5' LTRs suggests that CpG methylation had already silenced HERV-K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG methylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.

FIG. 1.

Transcriptional activities of various HERV-K(HML-2) 5′ LTRs in Tera-1 (left) and T47D (right) cells, as determined by luciferase reporter gene assays. LTR activities are given as percentages of a positive control vector driven by a CMV promoter. Standard deviations are indicated for each LTR. basic, a negative control vector that lacked a promoter sequence. All tested LTRs were inactive in T47D cells, shown on the right, solely to illustrate this finding.

FIG. 2.

CpG site methylation status of U3 regions of five HERV-K(HML-2) proviral 5′ LTRs in Tera-1 cells, as determined by bisulfite sequencing. The upper part of the figure depicts the examined LTR U3 region, including PCR primer locations for amplification of LTRs from the human genome. Primers FP2 were specific for flanking cellular sequences, while primer RP2 was specific for the LTR sequence. The different proviral 5′ LTRs examined are indicated on the left. Methylated CpG sites are indicated by solid circles, and unmethylated CpG sites are indicated by open circles. Thus, LTR-c7L and LTR-c6 are unmethylated, while the others are partially methylated. Note that methylation patterns for the latter LTRs are variable. Methylation levels for CpG sites as well as other C nucleotides are summarized as percentages on the right side of the figure. Positive and negative RT-PCR results indicate transcriptional activity of a particular provirus in Tera-1 cells, and fractional amounts of RT-PCR products from respective proviruses, as determined previously (28), are given in parentheses.

FIG. 3.

Transcriptional activity of in vitro-methylated HERV-K(HML-2) 5′ LTRs in Tera-1 cells. (A) Different methylation levels of in vitro-methylated reporter constructs. Shown here are in vitro methylation reactions of reporter plasmids that were terminated after 60 and 240 min. Plasmid DNAs were subsequently treated with methylation-sensitive BstUI restriction enzyme, and the fragments were electrophoresed. Note that BstUI partially digests plasmid DNA incubated for 60 min while plasmid incubated for 240 min is completely protected, indicating incomplete and complete methylation levels, respectively.-SAM, control reaction mixtures incubated for similar time periods but lacking SAM substrate. Thus, BstUI is able to completely digest these plasmid DNAs. (B) Effect of different CpG methylation levels on transcriptional activity of HERV-K(HML-2) 5′ LTRs. Three different LTRs with transcriptional activity were examined. Incubation times for in vitro methylation reactions are given in minutes. Transcriptional activities are given as percentages of a CMV-driven positive control vector. basic, a promoter-lacking negative control vector.

FIG. 4.

Occurrence of CpG sites in 5′ LTRs belonging to the HERV-K(HML-2) and the HERV-K(OLD) families (see the text for details). HS consensus is a previously reported consensus sequence for human-specific HERV-K(HML-2) LTRs (7). The entire 5′ LTR sequence is shown for each LTR. Each sequence was analyzed for the presence of CpG sites (triangles), as well as apparently mutated CpG sites (X's). TATA boxes are furthermore indicated for each LTR, when they could be identified. For each LTR, the 5′-most 620 bp were analyzed. Note that HERV-K(OLD) 5′ LTRs frequently display mutated CpG sites, indicating former CpG methylation.

FIG. 5.

CpG site methylation status of U3 regions of three HERV-K(HML-2) proviral 5′ LTRs in T47D cells, as determined by bisulfite sequencing. Please refer to the legend to Fig. 2 for details.