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DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis

Abstract

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

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Figure 1: WGS identifies a recurrent intronic mutation as the cause of XLPDR.
Figure 2: XLPDR is due to an intronic mutation that disrupts POLA1 expression.
Figure 3: POLA1 deficiency results in type-I interferon activation.
Figure 4: POLA1 deficiency leads to excessive activation of the IRF and NF-κB pathways.
Figure 5: POLA1 deficiency is associated with reduced levels of cytosolic RNA:DNA.
Figure 6: Cytosolic RNA:DNA attenuates the activation of IRFs.
Figure 7: The generation of cytosolic RNA:DNA via POLA1 modulates nucleic acid–sensor pathways.

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Acknowledgements

We thank the patients and their families for their participation in this research project and, in particular, the XLPDR International Association for support for the study of this disease; T. Hyatt for technical assistance with allelic discrimination assays; D. Baltimore (Caltech) for the lentiviral packaging plasmids pHCMV-VSV-G, pMDLg/pRRE and pRSV-Rev; Y. Pavlov (University of Nebraska) for the plasmid pcDNA3-POLA1; L. Schmitz (Justus-Liebig-University) for the HeLa fluorescent ubiquitination-based cell cycle indicator; J. Shay (University of Texas Southwestern Medical Center) for the gene encoding human telomerase; and H. Hobbs, J. Cohen, M. Attanasio and Z. (J.) Chen for comments and suggestions. Supported by the US National Institutes of Health (R01DK073639 to Ez.B.; R56AI113274 to Ez.B. and A.R.Z.; UL1TR001105 to J.J.R.; T32AI005284 to V.P.; R01AI098569 to N.Y.; and P30CA142543 for the UT Southwestern Live Cell Imaging Facility), the Children's Medical Center Foundation (A.R.Z.) and the National Natural Science Foundation of China (81271744 to Y.Y.).

Author information

Authors and Affiliations

Authors

Contributions

P.S. performed most of the cellular and biochemical experiments; T.G. analyzed human DNA samples and performed splicing studies; J.J.R. and C.X. analyzed WGS data; R.C.W. obtained skin biopsies and assisted with the hTERT immortalization of fibroblasts; H.L., S.K., N.M., P.R. and K.K. did some biochemical analysis; V.P. and N.Y. assisted with experiments on knockout mouse embryonic fibroblast cell lines; I.D. analyzed RNA-sequencing data; G.F., Zh.X., Zi.X., L.M., Z.L., H.W., Y.Y., D.B.-A., N.O., H.M., Eu.B., G.T., E.G. and A.S. contributed subjects; E.K.W. assisted with the analysis of cytoplasmic nucleic acids; M.T.d.I.M. performed clinical immunologic studies; A.R.Z. oversaw human genetic and splicing studies; Ez.B. oversaw cellular and biochemical studies; and P.S., A.R.Z. and Ez.B. wrote the manuscript.

Corresponding authors

Correspondence to Andrew R Zinn or Ezra Burstein.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Clinical characteristics of patients with XLPDR.

(a) Photographs of the new probands depicting the typical pigmentary changes as well as facial features that are characteristic of XLPDR. (b) Histopathology depicting skin changes. Black arrows point to melanophage accumulation in the dermis; open arrows point to accumulation of amyloid. (c) A representative example of the allelic discrimination assay for the POLA1 intronic variant in XLPDR probands, heterozygous carriers, and unaffected subjects from the Dallas Heart Study. (d) Immunoblotting for POLA1 and β-actin in lymphoblastoid cell lines. (e) Quantitative RT-PCR analysis of POLA1 mRNA in lymphoblastoid cell lines with normalization to ACTB. The probands analyzed are indicated in Fig. 1. (f) Densitometry-based quantification analysis of POLA1 protein levels in lymphoblastoid cell lines from (d) with normalization to β-actin. Data are pooled from 1 independent experiment (c), or representative from 2 (e,f) or 6 (d) independent experiments (individual values (c) or mean and individual sample values (e,f)).

Supplementary Figure 2 POLA1 deficiency does not affect cell proliferation.

(a) Proliferation rate of primary fibroblasts derived from an unaffected individual (WT, F1) and his XLPDR-affected son (P1). (b) Flow cytometric analysis of cell cycle distribution in XLPDR primary fibroblasts using propidium iodide staining. Numbers indicate the proportions of cells in G1, S, G2, and M stages.(c) Mitogen-activated lymphocyte proliferation was determined in XLPDR patients (n=2) and compared to unrelated healthy controls (n=2). (d) Flow cytometric analysis of cell cycle distribution in HeLa FUCCI cells transfected with control or POLA1 siRNA. Using the stably-expressed cell cycle fluorescent markers, the proportion of cells in G1, S, G2, and M stages was determined. POLA1 expression was determined by immunoblotting and normalized to β-actin (bottom panel). (e) RT-PCR for POLA1 mRNA to detect WT and aberrant transcripts using primer pairs A+B and X+B (see Fig. 2d) in LCL samples from patients and controls. The amplified products were analyzed by agarose electrophoresis and ethidium bromide staining. (f) Immunoblotting analysis of XLPDR cell lysates with four different commercial anti-POLA1. The specific epitopes of each antibody are depicted as well. Data are pooled from 3 (a), 2 (b,d,e) or 1 (c,f) independent experiments (mean and s.e.m.(a,c), individual sample values (b,d)).

Supplementary Figure 3 POLA1 deficiency results in increased responsiveness to diverse immunological stimuli.

(a) Quantitative RT-PCR analysis of ISG activation following in XLPDR-derived fibroblasts and normal controls following stimulation with various ligands, as indicated. For activation of dsDNA cytosolic sensors, cells were transfected with 1 μg/mL poly(dA:dT) for 16 h, or 2 μg/mL ISD or HSV-60 for 24 h. For dsRNA sensors, cells were transfected with 1 μg/mL poly(I:C) for 16 h (cyto poly(I:C)), or 0.5 μg/mL 5'-ppp-dsRNA for 24 h. To activate NF-κB-dependent gene expression, cells were stimulated with TNF (1000 U/mL) or IL-1β (1 μg/mL) for 0, 2, and 12 hours. To activte TLRs, cells were stimulated by adding to the culture media the following TLR agonists: poly(I:C) (250 μg/mL) for 2 and 7 hours, Pam3CSK4 (250 ng/mL) for 24 h, or LPS (10 μg/mL) for 24 h. (b) Quantitative RT-PCR analysis of ISG activation in cells with induced POLA1 deficiency following siRNA. After 48 h, cells were stimulated similarly to (a). Data are pooled from 4 independent experiments (a,b) (mean and s.e.m.(a,b)).

Supplementary Figure 4 Analyses of autoantibody production and leukocyte ultrastructure in patients with XLPDR.

(a) ELISA for anti-nuclear antibodies (ANA) in plasma from unaffected individuals (WT, n=16) and XLPDR probands (XLPDR, n=6). (b) Heatmap of the result of the large screen on presence of DNA-, RNA-, and protein-specific IgG and IgM autoantibodies in plasma of unaffected control individuals (WT, n=16) and XLPDR patients (XLPDR, n=6). The first two rows depict positive controls (total and control IgG or IgM, correspondingly). Full list of antigens is presented in Supplementary Table 4. Blue – low abundance, red – high abundance. (c) Electron microscopy of leukocytes from an XLPDR patient and a WT control. Magnification bar – 2 μM. *p=0.102 (unpaired Student's t-test). Experiments a-c were performed one time (mean and individual sample values (a), individual values (b)).

Supplementary Figure 5 Effects of POLA1 deficiency on the NF-κB pathway.

(a) Immunofluorescence analysis of TNF-induced nuclear accumulation of RelA (red) in XLPDR fibroblasts. Scale bar – 10 m. (b) Immunoblotting analysis of TNF-induced nuclear accumulation of RelA in XLPDR fibroblasts. (c) Immunoblotting analysis of total and phosphorylated forms of RelA in POLA1-deficient cells after TNF stimulation (left panel). Quantification of relative levels of two indicated forms of phospho-RelA was performed by quantitative fluorescence imaging (Li-COR, right panels). Data are representative from 2 (a) or 1 (b,c) independent experiment (individual values in (c)).

Supplementary Figure 6 Detection of cytosolic RNA:DNA.

(a) For the experiment shown in Fig. 5c, immunoblotting of the cytosolic and nuclear markers (RelA and p84, respectively) is shown (input fractions). In addition, the RNA:DNA immunoprecipitation was coupled to a control precipitation (RelA) to ensure equal lysate loading and bead preparation (IP). (b) Immunoprecipitation of RNA:DNA from three cell lines. Representative images of multiple beads stained with Picogreen from the experiment shown in Fig. 5c are shown here. Data are representative of 3 (a) and 4 (b) independent experiments.

Supplementary Figure 7 POLA1 localizes to the cytosol and co-localizes with RNA:DNA

(a) Immunofluorescence staining for POLA1 in the indicated cell lines. Cells were fixed and permeabilized with 100% methanol; POLA1 is visualized in green, and the nuclei were stained with DAPI. Scale bar – 10 μm. (b) Immunoblotting for POLA1 in nuclear and cytoplasmic fractions of the indicated cell lines. C - cytoplasm, N - nucleus. (c) Immunofluoresce staining for POLA1 (green) and RNA:DNA (red) in dermal fibroblasts. Scale bar – 10 μm. Red squares depict the original position of the enlarged area. Data are representative of 2 independent experiments (a,b,c).

Supplementary Figure 8 Quality control for the cell-fractionation procedure in Figure 6f.

Immunoblotting for POLA1 and the fraction marker proteins GAPDH (cytosol) and p84 (nucleus) was performed for the experiment shown in Fig. 6f. The experiment was performed one time.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 2, 5 and 6 (PDF 1533 kb)

Supplementary Table 1

Normalized gene expression data for RNA-seq experiments presented in Fig. 3b,c,d (XLSX 71 kb)

Supplementary Table 3

Summary table of clinical immunological data (XLSX 23 kb)

Supplementary Table 4

Extended data of screen on presence of IgG and IgM autoantibodies in plasma from XLPDR probands and unaffected indivduals, presented in Supplementary Fig. 4b (XLSX 132 kb)

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Starokadomskyy, P., Gemelli, T., Rios, J. et al. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis. Nat Immunol 17, 495–504 (2016). https://doi.org/10.1038/ni.3409

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