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DNA polymerase- α regulates type I interferon activation through cytosolic RNA:DNA synthesis

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting 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 expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response.

No MeSH data available.


POLA1 deficiency leads to excessive activation of IRF and NF-κB pathways(a) Immunoblot analysis of total and phosphorylated (p-) TBK1, and β-actin (loading control throughout) in lysates of wild-type and XLPDR fibroblasts left untreated (UT) or treated with poly(dA:dT) (16 h). (b) Immunoblot analysis of TBK1, IKKε and IRF3 (total and phosphorylated forms) in cytosolic (cyt) and nuclear (nucl) fractions of HeLa cells transfected with siRNA. When indicated, cells were treated with poly(dA:dT) as before. IκB-α and p84 serve as markers for the cytosolic and nuclear fractions, respectively. (c) Immunoblot analysis of total and p-RelA (on either S468 or S536), and IκB-α, in control and XLPDR-derived fibroblasts. When indicated, cells were treated with TNF. (d) Immunoblot analysis of RelA (total and phosphorylated forms) and IκB-α, in HeLa cells after POLA1 siRNA. When indicated, cells were treated with TNF. (e) Immunoblot analysis for the active form of the IKK complex (p-IKK1/2) after immunoprecipitation of the complex with a NEMO antibody. Control and XLPDR-derived fibroblasts were examined (left), as well as HeLa cells after POLA1 siRNA (right). When indicated, cells were treated with TNF. (f) qRT-PCR analysis of ISGs in wild-type (WT) mouse embryo fibroblasts (MEFs) after treatment with the TBK1 inhibitor (inh) BX795. *p<0.05 when compared to the control group (unpaired Student’s t test). Data are representative of 2 (a), 3(c,e,f) or 4 (b,d) independent experiments (mean and s.e.m.(f)).
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Figure 4: POLA1 deficiency leads to excessive activation of IRF and NF-κB pathways(a) Immunoblot analysis of total and phosphorylated (p-) TBK1, and β-actin (loading control throughout) in lysates of wild-type and XLPDR fibroblasts left untreated (UT) or treated with poly(dA:dT) (16 h). (b) Immunoblot analysis of TBK1, IKKε and IRF3 (total and phosphorylated forms) in cytosolic (cyt) and nuclear (nucl) fractions of HeLa cells transfected with siRNA. When indicated, cells were treated with poly(dA:dT) as before. IκB-α and p84 serve as markers for the cytosolic and nuclear fractions, respectively. (c) Immunoblot analysis of total and p-RelA (on either S468 or S536), and IκB-α, in control and XLPDR-derived fibroblasts. When indicated, cells were treated with TNF. (d) Immunoblot analysis of RelA (total and phosphorylated forms) and IκB-α, in HeLa cells after POLA1 siRNA. When indicated, cells were treated with TNF. (e) Immunoblot analysis for the active form of the IKK complex (p-IKK1/2) after immunoprecipitation of the complex with a NEMO antibody. Control and XLPDR-derived fibroblasts were examined (left), as well as HeLa cells after POLA1 siRNA (right). When indicated, cells were treated with TNF. (f) qRT-PCR analysis of ISGs in wild-type (WT) mouse embryo fibroblasts (MEFs) after treatment with the TBK1 inhibitor (inh) BX795. *p<0.05 when compared to the control group (unpaired Student’s t test). Data are representative of 2 (a), 3(c,e,f) or 4 (b,d) independent experiments (mean and s.e.m.(f)).

Mentions: Next, we examined whether signaling events that activate the IRF and NF-κB pathways are upregulated by POLA1 deficiency. In the IRF pathway, activation of various sensors results in the phosphorylation of the TBK1 and IKKε kinases, which in turn then phosphorylate IRF family members, promoting their dimerization and nuclear translocation35. Fibroblasts from XLPDR patients displayed robust TBK1 phosphorylation, even at baseline conditions, far exceeding the signature noted in wild-type cells (Fig. 4a). Similarly, silencing of POLA1 in HeLa cells led to phosphorylation of both TBK1 and IKKε, which exceeded that achieved in control cells after stimulation with poly(dA:dT). This signature was accompanied by enhanced phosphorylation of IRF3 in nuclear fractions (Fig. 4b). In the NF-κB pathway, we found that XLPDR fibroblasts achieved similar degrees of IκB degradation and nuclear NF-κB (RelA) accumulation after TNF stimulation (Fig. 4c and Supplementary Fig. 5a,b). However, in both fibroblasts and HeLa cells, POLA1 deficiency was associated with greater phosphorylation of RelA (Fig. 4c,d), which were also quantified using Li-COR (Supplementary Fig. 5c). This event is mediated by the IKK complex and can increase transcriptional induction36. In agreement with this finding, the phosphorylated and active forms of IKK1 and IKK2 were increased in patient fibroblasts as well as in HeLa cells after siRNA-mediated silencing of POLA1 (Fig. 4e). Thus, these data show that POLA1 deficiency leads to activation of signaling cascades that converge on TBK1, IKKε and the IKK complex. Blockade of TBK1 using a specific kinase inhibitor was sufficient to reverse the increased ISG expression (Fig. 4f), indicating that the activation of this kinase in XLPDR cells is necessary for the activation of IRF and its downstream gene targets. Altogether, these data indicate that POLA1 deficiency results in augmented signals converging on key kinases that activate IRF and NF-κB.


DNA polymerase- α regulates type I interferon activation through cytosolic RNA:DNA synthesis
POLA1 deficiency leads to excessive activation of IRF and NF-κB pathways(a) Immunoblot analysis of total and phosphorylated (p-) TBK1, and β-actin (loading control throughout) in lysates of wild-type and XLPDR fibroblasts left untreated (UT) or treated with poly(dA:dT) (16 h). (b) Immunoblot analysis of TBK1, IKKε and IRF3 (total and phosphorylated forms) in cytosolic (cyt) and nuclear (nucl) fractions of HeLa cells transfected with siRNA. When indicated, cells were treated with poly(dA:dT) as before. IκB-α and p84 serve as markers for the cytosolic and nuclear fractions, respectively. (c) Immunoblot analysis of total and p-RelA (on either S468 or S536), and IκB-α, in control and XLPDR-derived fibroblasts. When indicated, cells were treated with TNF. (d) Immunoblot analysis of RelA (total and phosphorylated forms) and IκB-α, in HeLa cells after POLA1 siRNA. When indicated, cells were treated with TNF. (e) Immunoblot analysis for the active form of the IKK complex (p-IKK1/2) after immunoprecipitation of the complex with a NEMO antibody. Control and XLPDR-derived fibroblasts were examined (left), as well as HeLa cells after POLA1 siRNA (right). When indicated, cells were treated with TNF. (f) qRT-PCR analysis of ISGs in wild-type (WT) mouse embryo fibroblasts (MEFs) after treatment with the TBK1 inhibitor (inh) BX795. *p<0.05 when compared to the control group (unpaired Student’s t test). Data are representative of 2 (a), 3(c,e,f) or 4 (b,d) independent experiments (mean and s.e.m.(f)).
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Figure 4: POLA1 deficiency leads to excessive activation of IRF and NF-κB pathways(a) Immunoblot analysis of total and phosphorylated (p-) TBK1, and β-actin (loading control throughout) in lysates of wild-type and XLPDR fibroblasts left untreated (UT) or treated with poly(dA:dT) (16 h). (b) Immunoblot analysis of TBK1, IKKε and IRF3 (total and phosphorylated forms) in cytosolic (cyt) and nuclear (nucl) fractions of HeLa cells transfected with siRNA. When indicated, cells were treated with poly(dA:dT) as before. IκB-α and p84 serve as markers for the cytosolic and nuclear fractions, respectively. (c) Immunoblot analysis of total and p-RelA (on either S468 or S536), and IκB-α, in control and XLPDR-derived fibroblasts. When indicated, cells were treated with TNF. (d) Immunoblot analysis of RelA (total and phosphorylated forms) and IκB-α, in HeLa cells after POLA1 siRNA. When indicated, cells were treated with TNF. (e) Immunoblot analysis for the active form of the IKK complex (p-IKK1/2) after immunoprecipitation of the complex with a NEMO antibody. Control and XLPDR-derived fibroblasts were examined (left), as well as HeLa cells after POLA1 siRNA (right). When indicated, cells were treated with TNF. (f) qRT-PCR analysis of ISGs in wild-type (WT) mouse embryo fibroblasts (MEFs) after treatment with the TBK1 inhibitor (inh) BX795. *p<0.05 when compared to the control group (unpaired Student’s t test). Data are representative of 2 (a), 3(c,e,f) or 4 (b,d) independent experiments (mean and s.e.m.(f)).
Mentions: Next, we examined whether signaling events that activate the IRF and NF-κB pathways are upregulated by POLA1 deficiency. In the IRF pathway, activation of various sensors results in the phosphorylation of the TBK1 and IKKε kinases, which in turn then phosphorylate IRF family members, promoting their dimerization and nuclear translocation35. Fibroblasts from XLPDR patients displayed robust TBK1 phosphorylation, even at baseline conditions, far exceeding the signature noted in wild-type cells (Fig. 4a). Similarly, silencing of POLA1 in HeLa cells led to phosphorylation of both TBK1 and IKKε, which exceeded that achieved in control cells after stimulation with poly(dA:dT). This signature was accompanied by enhanced phosphorylation of IRF3 in nuclear fractions (Fig. 4b). In the NF-κB pathway, we found that XLPDR fibroblasts achieved similar degrees of IκB degradation and nuclear NF-κB (RelA) accumulation after TNF stimulation (Fig. 4c and Supplementary Fig. 5a,b). However, in both fibroblasts and HeLa cells, POLA1 deficiency was associated with greater phosphorylation of RelA (Fig. 4c,d), which were also quantified using Li-COR (Supplementary Fig. 5c). This event is mediated by the IKK complex and can increase transcriptional induction36. In agreement with this finding, the phosphorylated and active forms of IKK1 and IKK2 were increased in patient fibroblasts as well as in HeLa cells after siRNA-mediated silencing of POLA1 (Fig. 4e). Thus, these data show that POLA1 deficiency leads to activation of signaling cascades that converge on TBK1, IKKε and the IKK complex. Blockade of TBK1 using a specific kinase inhibitor was sufficient to reverse the increased ISG expression (Fig. 4f), indicating that the activation of this kinase in XLPDR cells is necessary for the activation of IRF and its downstream gene targets. Altogether, these data indicate that POLA1 deficiency results in augmented signals converging on key kinases that activate IRF and NF-κB.

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting 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 expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-&alpha;. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response.

No MeSH data available.