SAMHD1 prevents autoimmunity by maintaining genome stability

Abstract

Objectives: The HIV restriction factor, SAMHD1 (SAM domain and HD domain-containing protein 1), is a triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs). Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory disorder that shares phenotypic similarity with systemic lupus erythematosus, including activation of antiviral type 1 interferon (IFN). To further define the pathomechanisms underlying autoimmunity in AGS due to SAMHD1 mutations, we investigated the physiological properties of SAMHD1.

Methods: Primary patient fibroblasts were examined for dNTP levels, proliferation, senescence, cell cycle progression and DNA damage. Genome-wide transcriptional profiles were generated by RNA sequencing. Interaction of SAMHD1 with cyclin A was assessed by coimmunoprecipitation and fluorescence cross-correlation spectroscopy. Cell cycle-dependent phosphorylation of SAMHD1 was examined in synchronised HeLa cells and using recombinant SAMHD1. SAMHD1 was knocked down by RNA interference.

Results: We show that increased dNTP pools due to SAMHD1 deficiency cause genome instability in fibroblasts of patients with AGS. Constitutive DNA damage signalling is associated with cell cycle delay, cellular senescence, and upregulation of IFN-stimulated genes. SAMHD1 is phosphorylated by cyclin A/cyclin-dependent kinase 1 in a cell cycle-dependent manner, and its level fluctuates during the cell cycle, with the lowest levels observed in G1/S phase. Knockdown of SAMHD1 by RNA interference recapitulates activation of DNA damage signalling and type 1 IFN activation.

Conclusions: SAMHD1 is required for genome integrity by maintaining balanced dNTP pools. dNTP imbalances due to SAMHD1 deficiency cause DNA damage, leading to intrinsic activation of IFN signalling. These findings establish a novel link between DNA damage signalling and innate immune activation in the pathogenesis of autoimmunity.

Keywords: Autoimmune Diseases; Autoimmunity; Fibroblasts; Systemic Lupus Erythematosus.

Figure 1

Dysregulation of deoxyribonucleoside triphosphate (dNTP) pools in SAMHD1-deficient fibroblasts causes genome instability leading to cell cycle delay and cellular senescence. (A, B) Fibroblasts from two patients with Aicardi–Goutières syndrome (AGS; AGS1, AGS2) exhibit markedly increased levels of deoxythymidine triphosphate (dTTP) and deoxyadenosine triphosphate (dATP) and proliferate more slowly than wild-type cells (WT, n=2). Shown are the means±SD of two independent experiments run in triplicate. (C) Synchronised AGS1 fibroblasts show delayed cell cycle progression, with an arrest in G₁ and S as well as (D) induction of the cell cycle inhibitors, p21 and p16, but not of p53 phosphorylated at S15. (E) Patient cells exhibit a senescent phenotype shown by a large increase in β-galactosidase-positive cells. Scale bar, 100 μm. (F) Alkaline single-cell gel electrophoresis reveals global DNA damage as shown by increased comet tail length and irregular nuclear contour in patient fibroblasts compared with wild-type cells. Scale bar, 10 μm. (G) Native patient cells do not show increased DNA double-strand breaks as determined by counting of γH2AX- and 53BP1-double positive nuclear foci, but are more susceptible to DNA double-strand breaks in response to low-dose ultraviolet (UV)-C (20 J/m²). Values represent means±SEM of two (C, F, G) or three (D, E) independent experiments run in triplicate. *p<0.05, **p<0.01, ***p<0.001 vs wild-type by Student's t test.

Figure 2

Genome-wide transcriptional profiles reveal upregulation of genes involved in DNA damage signalling and innate immune activation. (A) Heat maps based on RNA sequencing analysis represent hierarchically clustered transcripts showing up- or down-regulation by a factor of at least 2 in Aicardi–Goutières syndrome 1 (AGS1) and AGS2 compared with wild-type controls (WT, n=4). (B) Native patient fibroblasts exhibit constitutive upregulation of genes involved in the DNA damage response as well as (C) induction of interferon-stimulated genes. Indicated are the fold changes of the Reads Per Kilobase of exon per Million mapped reads (RPKM) values obtained by RNA sequencing analysis of patient fibroblasts (AGS1, AGS2) relative to the mean RPKM values of four WT cell lines.

Figure 3

SAMHD1 is regulated by cyclin A in a cell cycle-specific manner. (A) Coimmunoprecipitation with N- or C-terminally green fluorescent protein (GFP)-tagged SAMHD1 expressed in HEK293T cells immobilised on GFP-Trap beads pulls down cyclin A and cyclin-dependent kinase 1 (CDK1), but not cyclin E. (B) Reverse coimmunoprecipitation with two different cyclin A antibodies (Ab 1, Ab 2) confirms interaction of SAMHD1 and cyclin A at the endogenous level. (C) In vitro kinase assay reveals phosphorylation of SAMHD1 at T592 by cyclin A/CDK1, but not by cyclin A/CDK2 or cyclin E/CDK3. (D) Cyclin A and SAMHD1 colocalise within the nucleus as shown by a high correlation of fluorescence signals in the scatter plot. Scale bar, 10 μm. (E) Fluorescence cross-correlation spectroscopy of living HeLa cells cotransfected with mCherry–SAMHD1 (SAMHD1) and GFP–cyclin A (cyclin A) shows a high cross-correlation, indicating formation of mobile SAMHD1–cyclin A complexes. Compared with monomeric GFP or mCherry, the average normalised brightness of GFP–cyclin A and mCherry–SAMHD1 is 1 or 2, respectively. At least 15 cells were measured per experiment. Data represent means±SEM from two independent experiments. ***p<0.001 by Student's t test. (F) HeLa cells synchronised at G₀/G₁ were lysed at the indicated time points after serum re-addition (SR) and immunoblotted with the indicated antibodies. (G) Intracellular deoxyribonucleoside triphosphate (dNTP) concentrations in HeLa cells treated as in (F).

Figure 4

SAMHD1 knockdown recapitulates senescent phenotype and interferon (IFN) activation. (A) SAMHD1 is effectively knocked down in HeLa cells 72 h after transfection with SAMHD1-specific small interfering (si)RNAs (si-SAMHD1_1-3). Si-Ctrl, control-siRNA. This is accompanied by induction of p21. (B) Flow cytometry of propidium iodide-stained HeLa cells 72 h after transfection with si-SAMHD1_2 reveals delayed cell cycle progression and (C) an increase in β-galactosidase-positive cells. Scale bar, 10 μm. (D) SAMHD1 knockdown leads to induction of the IFNB gene and the interferon-stimulated genes IFI27, IFI6 and DDX58. Gene expression was normalised to GAPDH. Shown is the relative fold change in gene expression relative to control siRNA. Data are represented as mean±SEM from three (A) or four (B, C, D) independent experiments run in triplicate. *p<0.05; **p<0.01; ***p<0.001 by Student's t test.