SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters

Abstract

Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The majority of these repeats are retrotransposons of the SINE and LINE families, and such repeats are generally repressed by epigenetic mechanisms. It has been proposed that these elements can act as methylation centers from which DNA methylation spreads into gene promoters in cancer. Contradictory to a methylation center function, we have found that retrotransposons are enriched near promoter CpG islands that stay methylation-free in cancer. Clearly, it is important to determine which influence, if any, these repetitive elements have on nearby gene promoters. Using an in vitro system, we confirm here that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. SINE sequences themselves did not immediately acquire DNA methylation but were marked by H3K9me2 and H3K27me3. Moreover, our bisulfite sequencing data did not support that gain of DNA methylation in gene promoters occurred by methylation spreading from SINE B1 repeats. Genome-wide analysis of SINE repeats distribution showed that their enrichment is directly correlated with the presence of USF1, USF2, and CTCF binding, proteins with insulator function. In summary, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is counter-selected, except when the promoter is protected by an insulator element.

Fig. 1

Gene promoters with nearby SINE B1 show reduced transcriptional activity. (a) Plasmid constructs used to evaluate the effect of SINE B1 elements on promoter activity. The luciferase reporter gene was put under the control of mouse Cdkn2d, Mlh1 or p14Arf promoter, and SINE B1 elements were inserted in tandem 2 or 4 times immediately upstream to these promoters. B1a and B1b denote individual sequences with 19.2% and 4.9% divergence from the consensus SINE B1, respectively. (b) Transcriptional activity of Cdkn2d, Mlh1 and p14Arf promoters without (wt) and with SINE B1 insertions (2B1 and 4B1) in mouse NIH3T3 after transient transfection. The measured luciferase activity was reduced in SINE B1-containing plasmids compared to wt.

Fig. 2

SINE B1 elements cause transcriptional repression and epigenetic reprogramming of the p14Arf gene promoter. (a) Promoter activity over time with and without proximal insertion of SINE B1 elements. The p14Arf promoter show gradual decline in promoter activity only when SINE B1s are present. The results of two independent experiments are shown. Luciferase units were normalized by total protein in cell lysates. (b) De novo methylation of gene promoter. Compatible with the decrease in activity, the promoter of p14Arf show increased DNA methylation when SINE B1 elements are present. The methylation measurement was done by COBRA; the lower molecular weight band is resultant of cleavage of DNA when an internal CpG site to a selected restriction enzyme recognition site is maintained after bisulfite treatment, thus present only in amplicons originated from a methylated template. SssI is a positive control for presence of methylation and was generated by treating the plasmid DNA with the SssI methylase enzyme. Vector DNA is used as negative control of methylation. (c) Pyrograms showing the methylation status of CpG sites at positions +79bp to +88bp from TSS of the p14Arf promoter, 29 days after stable transfection in NIH-3T3 cells. PyroMeth is a highly quantitative method with better accuracy than bisulfite sequencing and COBRA. Note that 4B1-p14Arf and 6B1-p14Arf are hypermethylated, while wt-p14Arf and 2B1-p14Arf show low levels of DNA methylation. A graphic representation of pyroMeth analysis of nine CpG sites from positions -21bp to +88bp from TSS of the p14Arf promoter measured at 29 days after stable transfection is shown in the lower panel. (d) Switch of epigenetic marking. After initial strong DNA methylation, the p14Arf promoter with proximal SINE B1 elements lost this mark but stayed in a closed chromatin configuration, as indicated by the lack of H3K4me3 and H3K9ac as measured by ChIP assays.

Fig. 3

Mlh1 is refractory to silencing by SINE B1s. (a) As observed for p14Arf, the Cdkn2d promoter was sensitive to the presence of SINE B1s and showed gradual decline in promoter activity along time, but relatively stable activity when cloned in a B1-free reporter vector. (b) Increased DNA methylation density of the Cdkn2d promoter is correlated with the repression by SINE B1s. The gel image was captured as described in the Methods section from a COBRA experiment. SssI is a positive control for presence of methylation and was generated by treating the plasmid DNA with the SssI methylase enzyme. Vector DNA is used as negative control of methylation. (c) Mlh1 promoter is resistant to transcriptional silencing by SINE B1 retrotransposons. The Mlh1 promoter did not show an additional loss in promoter activity when cloned close to B1 elements, as measured by the luciferase assay. We inverted the direction of the Mlh1 promoter in the pGL3-basic plasmid (lower panel) to test whether the resistance to silencing by SINE B1 retrotransposons was dependent on its orientation. Similarly to the Mlh1 promoter cloned in positive orientation (i.e. the intronic region close to the luciferase gene), the Mlh1 promoter cloned in negative orientation with nearby B1 insertions did not show lower levels of expression compared to the retrotransposon-free plasmids. (d) In concordance with the maintained active status of the Mlh1 promoter, there was no gain in DNA methylation near the gene TSS as measured by COBRA. (e) Bisulfite sequencing of the Mlh1 promoter confirmed that the 4B1-Mlh1 plasmids did not gain methylation more rapidly than the wt-Mlh1 plasmids. Black circles represent methylated cytosines in a CpG dinucleotide, and empty circles are unmethylated cytosines. The average methylation of each region of the bisulfite sequenced Mlh1 sequence (upstream to TSS, 5′ UTR, coding region of the exon 1 and intron 1) is shown in the lower panel. Data from 90 days after transfection of both wt-Mlh1 and 4B1-Mlh1 were averaged. The 5′UTR is resistant to de novo methylation, compared to the other regions.

Fig. 4

The p14Arf promoter has a cryptic methylation center. (a) Four CpG sites in the p14Arf promoters gained methylation at a faster rate than other CpG sites at 22 days after transfection, and spreading of DNA methylation can be observed in the vicinity of these sites at days 36 and 50. The absence of methylation in the most 5′ CpG sites suggests that SINE B1 repeats did not function as methylation centers, and it is confirmed in (5a). Black circles represent methylated cytosines in a CpG dinucleotide, and empty circles are unmethylated cytosines. Each row of circles is an individual plasmid. (b) Average methylation per region. The p14Arf promoter was divided in four regions: the TSS+Exon, from the transcription start site to the end of the exon 1 according to the annotated gene CDS; Methylation Center, the region that first gained methylation in both wt- and B1-containing plasmids; Upstream to TSS and 5′ Distal, representing the portion of the sequenced region between the TSS+Exon 1 and the Methylation Center, and upstream to the Methylation Center, respectively. Methylation density is shown for each of these for regions as measured on day 50 by bisulfite sequencing.

Fig. 5

SINE B1 elements remained methylation-free and do not function as methylation centers in this system. (a) Bisulfite sequencing of B1 sequences upstream to the p14Arf promoter shows no sign of significant cytosine methylation of these elements. (b) Despite their DNA methylation-free status, SINE B1s (p14Arf-B1 columns) are targeted by chromatin-remodeling complexes and become marked by H3K9me2 and H3K27me3. tuba is an active gene and was used as positive control for H3K4me3 and H3K9ac; Hbb-b1 and Nanog are repressed in mouse NIH/3T3 cells and were used as positive controls for H3K9me2 and H3K27me3.

Fig. 6

The human CDH1 gene promoter is sensitive to SINE, but not LINE retrotransposons. (a) Cloning strategy of the CDH1 gene promoter in the pGL3-basic vectors, with and without SINE and LINE retrotransposons. As shown, for different constructs were transiently transfected in RKO (colon cancer cell line) and NCI-H1299 (lung cancer cell line). (b) Promoter activity of different CDH1 constructs 36 hours after transfection. Similarly to mouse B1 elements, human Alu (a) and Mir retrotransposons (m) were associated with lower transcriptional activity of the CDH1 gene promoter in RKO cells, while LINE sequence (L2) did not cause change in gene expression. This pattern was not reproducible in NCI-H1299 cells, suggesting that cellular mechanisms interfere with the capacity of SINEs in promoting gene repression

Fig. 7

SINE retrotransposons accumulate near gene promoters bound by insulator proteins. Gene promoters were grouped according their overlap with CpG islands (CGI) and binding by CTCF (mouse and human promoters), USF1 and USF2 (human promoters only). The graphics show the frequency of SINE repeats in a 20-kb window centered in genes TSS. Except for promoters CGI bound by CTCF, the presence of an insulator created an environment that is more permissive to SINE repeats than insulator-free promoters.