SAMHD1 restricts the deoxyguanosine triphosphate pool contributing to telomere stability in telomerase-positive cells

Abstract

SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.

Keywords: Aicardi-Goutières syndrome (AGS); deoxynucleotide metabolism; deoxynucleotide pool imbalance; sterile alpha motif and HD domain containing protein 1 (SAMHD1); telomere maintenance.

FIGURE 1

hTERT immortalized fibroblasts retain the distinctive dNTP pool content of the corresponding primary cells. (A) Cumulative population doublings (PDs) curves of WT (WT1 and WT2) and AGS (P1 and P2) fibroblasts after immortalization with hTERT. Viable cells were counted every 4 days for about seven months. The daily population doubling numbers (PDN) were calculated by the formula {ln [(number of cells harvested)/number of cell seeded)]/ln2}/4 days and added to the previous PDNs to yield the cumulative PDN value. (B) Relative telomerase activity was measured by TAQ PCR at Mid stage in two biological samples of each immortalized fibroblast line analyzed in triplicate. Telomerase activity data are relative to the activity measured in hTERT WT1 fibroblasts. In all primary cell lines telomerase activity was undetectable or <10⁻⁵ relative to WT1. Bars represent means ± SE. (C) Fold change in the dNTP pool concentrations relative to WT immortalized fibroblasts (WT1 + WT2) at Early stage after hTERT immortalization. dNTP pools were measured in at least three biological samples analyzed in duplicate. Bars represent means ± SE. Statistical analysis was carried out by comparing the pooled data from WT (WT1 + WT2) and AGS cells (P1 and P2) using the Student's t test (two‐tailed); ***p < .001. (D) Proportion of each dNTP as a percentage of the total dNTP pool in cycling WT (WT1 and WT2) and AGS fibroblasts P1 and P2.

FIGURE 2

SAMHD1 deficiency promotes telomere over‐elongation in hTERT immortalized AGS fibroblasts. (A) Mean telomere lengths measured by qPCR in primary AGS (P1 and P2) and WT (WT1 and WT2) fibroblasts at early (˂15) passages from isolation and in Early, Mid and Late samples after hTERT immortalization. Genomic DNA sample with known telomere length was used as a reference. Bars represent means ± SE of two biological replicates for each fibroblast line, each analyzed in triplicate. ***p < .001 (two‐tailed Student's t test on pooled data from WT and AGS cells). (B) Telomere length measured using metaphase Q‐FISH in Early, Mid and Late samples of hTERT AGS P1 and P2 and WT fibroblasts (WT1 and WT2). At least 50 metaphases were analyzed for each cell line and for each stage (Early, Mid and Late) indicated in (A). Telomere length is calculated as the ratio of telomere/centromere fluorescence intensity. Bars represent means ± SE. Statistical analyses as described in A, with ***p < .001. No significant difference in the number of telomeric ends lacking a fluorescence signal was observed in any of these cell populations.

FIGURE 3

SAMHD1 deficiency promotes telomere over‐elongation in hTERT‐positive cancer cells. (A) Fold change in the dNTP pool concentrations in SAMHD1‐KO SUIT2 cells relative to SAMHD1‐proficient SUIT2 cells. dNTP pool sizes were measured in cycling cultures of SUIT2 and SAMHD1‐KO SUIT2 cells with similar percentages of S phase cells (30‐35%), by a DNA polymerase‐based assay. Data are means from four biological samples analyzed in duplicate. Bars represent means ± SE. **p < .01 and ***p < .001 (two‐tailed Student's t test). (B) Proportion of each dNTP as a percentage of the total dNTP pool in SUIT2 WT and SAMHD1 KO cells. (C) Telomerase activity in SAMHD1‐KO and parental SAMHD1‐proficient cancer cells. Telomerase activity was measured by TAQ PCR in SAMHD1‐KO THP1 and SAMHD1‐KO SUIT2 and normalized to telomerase activity in SAMHD1 proficient THP1 and SUIT2 cells respectively. Data are means of two biological samples for each cell line, each analyzed in triplicate. Bars represent means ± SE. *p < .05 and ***p < .001 (two‐tailed Student's t test). (D) Mean telomere lengths measured by qPCR analysis in SAMHD1‐KO THP1 and SAMHD1‐KO SUIT2 cells relative to SAMHD1 proficient THP1 and SUIT2 cells, respectively. Bars represent means ± SE. ***p < .001 (two‐tailed Student's t test).

FIGURE 4

Telomere over‐elongation in TetR‐shSAMHD1 SUIT2 cells depends on prolonged SAMHD1 depletion. (A) SAMHD1 expression in tetR‐shSAMHD1 SUIT2 cells during prolonged silencing with doxycycline. SAMHD1 mRNA expression was analyzed in tetR‐shSAMHD1 SUIT2 cells by qRT‐PCR and normalized to Succinate Dehydrogenase Complex Flavoprotein subunit A (SDHA) mRNA expression. The abundance of SAMHD1 was calculated relative to the expression detected in SAMHD1 proficient SUIT2 cells. TetR‐shSAMHD1 SUIT2 cells were cultured for 1, 2 and 3 months in either the presence or absence of 50 ng/mL doxycycline. Data are means of two independent experiments for each stage analyzed in triplicate. Bars represent means ± SE. ***p < .001 (two‐tailed Student's t test). (B) dNTP pool content was measured in TetR‐shSAMHD1 SUIT2 cells kept in culture for two months in either the presence or absence of 50 ng/mL Doxycycline. Data were obtained from two biological samples of each cell line, each analyzed in triplicate. Bars represent means ± SE. ***p < .001 (two‐tailed Student's t test). (C) Proportion of each dNTP as a percentage of the total dNTP pool in tetR‐shSAMHD1 SUIT2 cells cultured in the presence or absence of 50 ng/mL doxycycline. (D) Mean telomere lengths measured by qPCR analysis in tetR‐shSAMHD1 SUIT2 cells relative to SAMHD1 proficient SUIT2 cells. TetR‐shSAMHD1 SUIT2 cells were cultured for 1, 2 and 3 months in either the presence or absence of 50 ng/mL doxycycline. Bars represent means ± SE. ***p < .001 (two‐tailed Student's t test).

FIGURE 5

SAMHD1 is required to suppress telomere fragility in hTERT immortalized skin fibroblasts. Representative images of a normal chromosome (A) and two chromosomes with fragile telomeres indicated by white arrows in (B). Telomeres and centromeres were detected respectively with Alexafluor‐488 and Alexafluor‐647, chromosomes were counterstained with DAPI. (C) Quantification of fragile telomeres per metaphase in Early, Mid and Late samples of hTERT AGS P1 and P2 and WT fibroblasts (WT1 and WT2) analyzed by metaphase Q‐FISH. Proportions were calculated from at least 20 metaphase spreads for each cell line and stage. Bars represent means ± SE. ***p < .001 (two‐tailed Student's t test, as applied for mean comparisons, or Analysis of variance as applied on the whole set of data).

FIGURE 6

A Model for SAMHD1 as a gatekeeper of telomere stability in telomerase positive cells. In SAMHD1 proficient cells the dGTP pool represents less than 10% of the total dNTP pool which preserves telomere length and structure. SAMHD1 deficiency both in immortalized fibroblasts and telomerase positive cancer cells expands the dGTP content which enhances telomerase processivity leading to telomere elongation (open arrows) and fragility (dark arrows).