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. 2017 Jun;1(1):2.
doi: 10.3390/epigenomes1010002. Epub 2016 Sep 22.

Helicase Lymphoid-Specific Enzyme Contributes to the Maintenance of Methylation of SST1 Pericentromeric Repeats That Are Frequently Demethylated in Colon Cancer and Associate with Genomic Damage

Johanna K Samuelsson  1   2 , Gabrijela Dumbovic  3 , Cristian Polo  3 , Cristina Moreta  3 , Andreu Alibés  3 , Tatiana Ruiz-Larroya  1 , Pepita Giménez-Bonafé  4 , Sergio Alonso  3 , Sonia-V Forcales  3 , Perucho Manuel  1   3   5
Affiliations

Helicase Lymphoid-Specific Enzyme Contributes to the Maintenance of Methylation of SST1 Pericentromeric Repeats That Are Frequently Demethylated in Colon Cancer and Associate with Genomic Damage

Johanna K Samuelsson et al. Epigenomes. 2017 Jun.

Abstract

DNA hypomethylation at repetitive elements accounts for the genome-wide DNA hypomethylation common in cancer, including colorectal cancer (CRC). We identified a pericentromeric repeat element called SST1 frequently hypomethylated (>5% demethylation compared with matched normal tissue) in several cancers, including 28 of 128 (22%) CRCs. SST1 somatic demethylation associated with genome damage, especially in tumors with wild-type TP53. Seven percent of the 128 CRCs exhibited a higher ("severe") level of demethylation (≥10%) that co-occurred with TP53 mutations. SST1 demethylation correlated with distinct histone marks in CRC cell lines and primary tumors: demethylated SST1 associated with high levels of the repressive histone 3 lysine 27 trimethylation (H3K27me3) mark and lower levels of histone 3 lysine 9 trimethylation (H3K9me3). Furthermore, induced demethylation of SST1 by 5-aza-dC led to increased H3K27me3 and reduced H3K9me3. Thus, in some CRCs, SST1 demethylation reflects an epigenetic reprogramming associated with changes in chromatin structure that may affect chromosomal integrity. The chromatin remodeler factor, the helicase lymphoid-specific (HELLS) enzyme, called the "epigenetic guardian of repetitive elements", interacted with SST1 as shown by chromatin immunoprecipitation, and down-regulation of HELLS by shRNA resulted in demethylation of SST1 in vitro. Altogether these results suggest that HELLS contributes to SST1 methylation maintenance. Alterations in HELLS recruitment and function could contribute to the somatic demethylation of SST1 repeat elements undergone before and/or during CRC pathogenesis.

Keywords: DNA demethylation; colorectal cancer; epigenetics; satellite element.

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Conflict of interest statement

Conflicts of Interest: The authors declare that they have no conflicts of interest.

Figures

Figure 1.
Figure 1.
SST1 elements are demethylated in colorectal cancer. (A) DNA fingerprinting by methylation sensitive-amplified fragment length polymorphism (MS-AFLP) identified a hypomethylated region in 22% of colorectal cancer (CRC) tumors (band C-5). The insert depicts the region of the fingerprint of two CRC cases, showing the intense bandC-5 in tumor 153, indicating demethylation of the NotI site. Band C-5 correspond (bed to a pericentromeric region on chromosome 21, which contains 21 SST1 satellite tandem repeats. Most of these SST1 elements contain a NotI site; that can be detected by MS-AFLP. Each repeat of approximately 1.3 kb (grey rectangles) is flanked by CA- and GA-rich regions. On each side of the21 repeat region, families of retrotransposons are present (MIRC and LIM2). N and T: normal and tumor tissue DNA; (B) A region of 317 bp comprising 28 CpG sites and the NotI site was analyzed by bisulfite sequencing in several CRCs. Analysis of case 153 is represented, where black and white circles represent methylated and unmethylated CpG sites, respectively. Small black dots indicate polymorphisms including TpA sequences possibly resulting from spontaneous deamination of methylated CpGs on the opposite DNA strand. Each row represents the sequence of an individual plasmid clone; and (C) methylation status of SST1 satellites from chromosome 21 in 12 CRCs cases. SST1 methylation levels indicate the methylation average of the 28 CpGs analyzed by bisulfite sequencing from individual PCR clones (rows in (B)) in colon normal samples (black) and tumor samples (white). Severe demethylated cases show a methylation average difference between normal and tumor equal or higher than 10%, whereas moderate demethylation cases show a difference between 5% and 10%.
Figure 2.
Figure 2.
Demethylation of SST1s is associated with genomic damage in CRCs with wild-type (WT) TP53, but not with mutant (MUT) TP53. p values were calculated by t test. Genomic damage fraction (GDF) was estimated by AP-PCR DNA fingerprinting as previously described [21]. GDF indicates average number of copy number changes (losses and gains) in tumor relative to normal tissue. NC: no change in DNA methylation; HYPO: DNA hypomethylation.
Figure 3.
Figure 3.
SST1 methylation and clinicopathological features of CRC. CRC cases with quantitative information of SST1 somatic demethylation (n = 80) were classified into two groups, i.e., below (green lines) and above (blue lines) increasing SST1 demethylation cutoff values. We explored the effect of the different cutoffs between −5% and 22%, with a 0.1% increment. The information of every clinicopathological and mutational factor is represented in a stacked pair of graphs. The upper graph of every pair shows, in a negative logarithmic scale (y axis), the p value of the comparison between tumors below and tumors above the SST1 demethylation cutoff employed for the classification (x axis). In pink, p values < 0.05. In red, p values < 0.01. The lower graph of every pair shows the values of the parameters (y axis) for the group of tumors below (green) or above the cutoff (blue). The SST1 demethylation regions where the applied cutoff yielded statistical significance are also indicated in pink (p < 0.05) and red (p < 0.01). Age was compared by Student’s t test. Gender, KRAS mutations and TP53 mutations were analyzed by Fisher’s exact test. Shaded in light blue, the region around the 10% demethylation cutoff.
Figure 4.
Figure 4.
SST1, but not SATα methylation, correlates positively with histone 3 lysine 9 trimethylation (H3K9me3) levels and negatively with histone 3 lysine 27 trimethylation (H3K27me3). (A) SST1 and SATα methylation levels of individual clones analyzed by bisufite sequencing in CIRC cell lines Caco-2 and LS174. As in Figure 1C, each dot corresponds to average methylation level present in one PCR clone sequence; (B) chromatin immunoprecipitation analysis (ChIP) of histone modifications on SST1 element. Demethylation of SST1 associates with increased H3K27me3 and reduction of H3K9me3 in SST1 chromatin. Demethylation of SATα element associates with decrease of H3K9me3 but not with H3K27me3 increase. Fold change was calculated relative to the input (comparative Ct method). IgG indicate immunoprecipitation background levels; (C) ChIP analysis of histone modifications associated with SST1 repetitive elements in two representative cases of colon cancer formalin-fixed paraffin-embedded (FFPE) primary tissue. Both tumors (709 and 726) displaying severe and moderate SST1 demethylation, respectively (Figure 1C), showed a shift from high levels of SST1 methylation and H3K9me3 enrichment in normal tissue (black bars) to SST1 demethylation accompanied with H3K9me3 reduction and H3K27me3 increase in the tumor (white bars); and (D) demethylated SST1 DNA associates with H3K27me3 by ChIP analysis in CRC cell line LS174T. The immunoprecipitated genomic DNA regions were bisulfite sequenced to monitor SST1 (ChIP-BS-seq). Input DNA: bisulfite treated input DNA purified after sonication. H3K27me3 DNA: bisulfite-treated DNA after H3K27me3 immunoprecipitation. SST1 elements associated with H3K27me3 show significantly higher demethylation compared to the input. p value was calculated by t test.
Figure 5.
Figure 5.
SST1 demethylation changes histone modifications in CRC] cell line HCT116. (A) SST1 methylation levels of individual clones estimated by bisulfite sequencing. Untreated (Unt) or treated with 5-aza-dC at 2.5 μM (AZA); and (B) SST1 demethylation, induced by 5-aza-dC (AZA) treatment (2.5 mM), is followed by an increase in H3K27me3 levels as shown by chromatin immunoprecipitation. H3K9me3 and H3K27me3 levels shown as fold enrichment relative to the input. IgG indicate immunoprecipitation background levels.
Figure 6.
Figure 6.
HELLS is recruited to SST1 regions, localizes in the centrosome and the mitotic spindle, and HELLS knockdown results in SST1 demethylation. (A) ChIP analysis of HELLS and DNA methyltransferase 3B (DNMT3B) recruitment to SST1 repetitive elements in CRC cell line HCT116 (left graph). The right graph shows HELLS recruitment to SST1 monitored by ChIP in Caco-2 (SST1 methylated) and LS174 (SST1 demethylated). Relative recruitment was calculated by the comparative Ct method and the input values were used as normalizers. IgG shows art immunoprecipiation background; (B) HELLS expression by confocal immunofluorescence. Caco-2 cells stained with primary antibody against HELLS (red) and counterstained with 4′,6-diamidino-2-phanylindole (DAPI) (blue). Image analysis by Slidebook software v4 (Intelligent Imaging Innovations, Denver, CO, USA) shows increased protein levels of HELLS around what seems to be the centrosome regions of mitotic cells; (C) Co-immunofluorescence of HELLS with α-tubulin, a marker of the mitotic spindle, in HCT116 cells; (D) Western blot on nuclear extracts from HCT116 polyclones obtained after lentiviral infection of small hairpin RNAs (shRNA) of a scrambled control sequence (shCtrl) or targeting HELLS (shHELLS); and (E) SST1 methylation levels of individual clones analyzed by bisulfite sequencing in colon cancer cell line HCT116 with scrambled shRNA (shCtrl) or shRNAs for HELLS (shHELLS). This analysis was performed three weeks post-infection. p value was calculated by Mann Whitney test.
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