Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island - PubMed (original) (raw)

Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island

Heather M O'Hagan et al. PLoS Genet. 2008.

Abstract

Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island-containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Treatment with tetracycline induces a double strand break in the inserted E-cad promoter.

(A) MDA-MB-231 cells were stably transfected with constructs expressing the tet repressor, the HA-tagged I-SceI enzyme, and the E-cadherin promoter containing the I-SceI consensus cut site driving the Herpes Simplex Virus thymidine kinase gene (HSVTK). The cell line used throughout this study containing these 3 vectors is named ROS8. (B) Time course for tet treatment. (C) Treatment of cells with 1 ug/ml tet induces expression of the HA-I-SceI enzyme by RT-PCR. Expression of HSVTK by RT-PCR remains unchanged. (D) At the 4+4 hour time point, the majority of the cells express the HA-I-SceI enzyme by immunofluorescence. Cells were fixed after treatment with tet as indicated. HA-I-SceI enzyme localization was determined using an anti-HA primary antibody followed by an anti-rabbit FITC secondary antibody (green). Blue = nuclear DAPI stain. (E) DNA was collected from cells treated with tet as indicated. PCR was performed using primers on either side of the cut site with the 3′ primer being unique for the exogenous E-cad promoter. (F) Cells treated with tet were analyzed via ChIP for the enrichment of phospho-H2AX using primers in the promoter region (labeled SCE) and using primers in the gene sequence (labeled TK). The average change in phospho-H2AX recruitment over input as measured by ChIP was quantitated by gel densitometry with error bars indicating the standard error of 3 PCRs.

Figure 2. DSB damage and/or repair induces the transient recruitment of SIRT1, DNMT1, and DNMT3B.

(A) Cells were treated with tet as indicated, crosslinked and sonicated for ChIP. A western blot for HA-I-SceI and phospho-H2AX was performed using a portion of the sonicated material. (B) SIRT1 is localized to the DNA in the vicinity of the cut site following DNA damage. ChIP was performed using the material from (A) and antibodies against SIRT1 and H4K16ac. Representative gels (left panel) are shown for PCR using the promoter SCE primers. Graphs (right panel) are shown, using primers for the SCE and TK regions, for the quantitative average change in recruitment over input as measured by gel densitometry. Error bars indicate the standard error for three or four PCRs. (C) Silent chromatin marks are observed transiently in the vicinity of the cut site. ChIP was performed using the material treated as in (A) and employing antibodies against di- and trimethyl H3K9 and H3K27me3. Data is presented as in (B). Error bars indicate the standard error for four PCRs. (D) DNMTs are localized to the chromatin near the cut site. ChIP was performed using the material treated as in (A) and employing antibodies against DNMT1 and DNMT3B. Data is presented as in (B). Error bars indicate the standard error for three PCRs.

Figure 3. Effects of knockdown of SIRT1 by siRNA.

(A) Cells were transiently transfected for three consecutive days with non-target siRNA (NT) or SIRT1 siRNA (SIRT1), treated with tetracycline to induce HA-I-SceI as indicated, and crosslinked and sonicated for ChIP analyses. A western blot was performed for anti-SIRT1, anti-acetyl lysine 382 of p53, anti-HA, and anti-β-actin using a portion of the sonicated sample. (B) Changes in ChIP results at the SCE site with SIRT1 knockdown. ChIP was performed using material from (A) and antibodies against SIRT1 and H4K16ac. Representative gels (top panels) are shown for PCR using primers in the SCE promoter region. The average change in recruitment to the promoter over input as measured by ChIP was quantitated (bottom panels) by gel densitometry for three PCRs with error bars indicating the standard error. (C) Changes in ChIP results at the TK site with SIRT1 knockdown. Using ChIP samples from (B) PCR was performed using primers in the body of the gene labeled TK with representative gels (top panels) and quantitation of results as in (B) (bottom panels). (D) Effects of knockdown of SIRT1 on phospho-H2AX recruitment kinetics. ChIP was performed using antibodies against phospho-H2AX. Representative gels (top panels) are shown for PCR using primers in the promoter region. Graphs for quantitation (bottom panel) are shown using the SCE primers, and error bars indicate the standard error for three PCRs. (E) Effects of knockdown of SIRT1 on the kinetics of break repair. Input DNA was used from the ChIP samples from (B). PCR was performed using primers on either side of the cut site with the 3′ primer being unique to the exogenous E-cad promoter. PCR using genomic GAPDH primers is used as a loading control. The average change in PCR across the break site over GAPDH was quantitated by gel densitometry for four PCRs with error bars indicating standard error (bottom panel).

Figure 4. Changes in enrichment of silencing proteins and chromatin marks with knockdown of SIRT1.

(A) Changes in ChIP results at the SCE site with SIRT1 knockdown. Using sonicated material from Figure 3A, ChIP was performed using antibodies against EZH2 and H3K27me3. Representative gels (top panels) are shown for PCR using primers in the SCE promoter region. The average change in recruitment to the promoter over input as measured by ChIP was quantitated (bottom panels) by gel densitometry for three PCRs with error bars indicating the standard error. (B) Changes in ChIP results at the TK site with SIRT1 knockdown. Using ChIP samples from (A), PCR was performed using primers in the body of the gene labeled TK with representative gels (top panels) and quantitation of results as in (A) (bottom panels). (C) DNMT3B localization to the cut site is lost when SIRT1 is knocked down. Cells were treated as in (A). ChIP was performed using antibodies against DNMT1 and DNMT3B. Representative gels are shown for PCR using primers in the SCE promoter region (top panels). The average change in recruitment to the promoter over input as measured by ChIP was quantitated by gel densitometry for three to four PCRs with error bars indicating the standard error (bottom panels). (D) Using ChIP samples from (C), PCR was performed using primers in the body of the gene labeled TK with representative gels (top panels) and quantitation of results as in (C) (bottom panels).

Figure 5. Inducing a DSB in a promoter can lead to silencing and the seeding of methylation.

(A) Cells were untreated (P) or treated for 4 hours (named A) or 24 hours (named B) with tetracycline. Silencing of HSVTK was then selected for by ganciclovir treatment. Clones that survived selection were analyzed by RTPCR for HSVTK and GAPDH expression. (B) Early passages of HSVTK silent clones have an enrichment of DNMT1, DNMT3B, EZH2 and silent chromatin marks at the SCE promoter. ChIP assays for all marks in the figure were performed on cells where no DSB was induced and no cell selection was initiated (P) and two HSVTK silent clones five passages after selection with ganciclovir (1B and 3B). Representative gels are shown for PCR using primers in the SCE promoter region. (C) Late passages (p34 to p36 after selection with ganciclovir) of HSVTK silent clones show enrichment for DNMT1 but not DNMT3B. ChIP was performed with cells as in (B). Representative gels are shown for PCR using primers in the SCE promoter region. (D) Bisulfite sequencing data for DNA methylation status of clones. DNA was isolated from uncut, unselected cells (parental) and two HSVTK silent clones (1B and 8B) at passages 1, 10, 20 and 30 after ganciclovir selection. Bisulfite sequencing was performed using primers on either side of the cut site with the 3′ primer being specific for the exogenous E-cad promoter. Open circles indicate unmethylated CpGs and closed circles indicate methylated CpGs. The location of the Sce cut site, the transcription start site, the SCE primers used for ChIP, and the well-characterized E-cad E-boxes (E1, E2, and E3) are indicated. (E) CpG methylation spreads with passage. The E-cad promoters containing the cut site were bisulfite sequenced in HSVTK silent clones at passages 1, 10, 20 and 30 after ganciclovir selection. A mean number of methylated CpGs per bisulfite sequenced clone is reported. A minimum of 6 bisulfite clones were sequenced per HSVTK silenced clone. The means presented are determined from the data shown in (D) plus additional unmethylated clone 6B and methylated clone 3B. (F) Effects of DAC and TSA treatment on expression of HSVTK as analyzed by realtime RT-PCR. In the lesser DNA methylated clone, 1B, both drugs lead to increased TK expression, while in the more DNA methylated clone, 8B (see panels D and E), DAC induces more increased expression than TSA treatment. Parent clones, or passage 30 of HSVTK silent clones, were treated with 1 µM deoxyazacytidine once a day for three days or once with 300 nM TSA for 16 hours. Realtime RT-PCR was performed for HSVTK expression. The mean HSVTK expression is shown in relation to expression in untreated parental cells with error bars indicating the standard error for three independent experiments.

Figure 6. Reduction of SIRT1 during DNA damage decreases the number of silent clones that have methylation.

(A) Cells were treated for three consecutive days with non-target (NT) or SIRT1 siRNA followed by 24 hours of tetracycline. Cells were then selected for silencing of the HSVTK gene by treatment with ganciclovir. DNA was isolated from clones that survived ganciclovir treatment. Bisulfite sequencing was performed as outlined in Figure 5. One representative bisulfite sequenced clone is presented for each HSVTK silent clone. (B) A dot plot of the mean number of CpGs methylated per HSVTK silenced clone from either un-siRNA treated cells (untreated) (Figure 5D & E), non-target siRNA treated cells (NT), or SIRT1 siRNA treated cells (SIRT1). The number of clones without methylation versus the number with methylation is significantly different between the SIRT1 knockout cells and the non-target cells (the asterisk indicates p<.05 by chi-square test). (C) The mean number of methylated CpGs per all HSVTK silent clones from either un-siRNA treated cells, NT siRNA treated cells, or SIRT1 siRNA treated cells. Error bars indicate the standard error. The difference in the mean for the SIRT1 siRNA treated cells is significantly different from that of the NT siRNA treated cells (the asterisk indicates p<.05 by Student's T-test). (D) Model for double strand break induced silencing of a gene. Initially after a DSB occurs in the promoter of a gene H2AX is phosphorylated (orange circles) and H4K16 is acetylated (green circles) causing the chromatin to open, allowing access to the break by repair factors and a stimulation of DNA damage signaling. Then SIRT1, EZH2, DNMT1, and DNMT3B are recruited to the area around the break site resulting in a decrease in H4K16ac and an increase in H3K27me3 (purple circles). These modifications result in compaction of the chromatin around the break site possibly causing a reduction in DNA damage signaling initiated by the prior decondensation of the chromatin or preventing transcription of unrepaired DNA. In the majority of cells (99.1%) the DNA is repaired and the chromatin returns to its original state. In a small fraction of the cells (0.9%) the promoter becomes silenced and gene expression is lost possibly due to the persistent localization of EZH2, DNMT1, and DNMT3B to the area of the break site and the prolonged condensed chromatin. Additionally, there is a seeding of DNA methylation in the area 3′ to the break site (white circles – unmethylated CpGs; black circles – methylated CpGs). After passage, DNMT3B is no longer localized to the promoter but EZH2 and DNMT1 are retained. The DNA methylation continues to spread further stabilizing the silencing of the downstream gene.

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Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island - PubMed (original) (raw)