Upregulated LINE-1 Activity in the Fanconi Anemia Cancer Susceptibility Syndrome Leads to Spontaneous Pro-inflammatory Cytokine Production

Abstract

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4(FANCP) deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis.

Keywords: Cytoplasmic DNA; DNA damage; Fanconi Anemia; Inflammation; Innate immune sensing; Interferon; SLX4 complex; cGAS-STING; endogenous retroelement.

Fig. 1

The SAP MUS81-EME1-interacting domain of SLX4 is required for repressing inflammatory signals. (A) IFNα, IFNβ, IFNγ and MxA mRNA levels were measured by RT-qPCR in RA3331 cells complemented either with WT-SLX4 (RA3331^SLX4) or SLX4ΔSAP (RA3331^SLX4ΔSAP). Graphs present means (± SD) of triplicate measurement of 6 independent experiments expressed as fold change in mRNA expression relative to RA3331^SLX4. ****: p < 0.001; ***: p < 0.005; **: p < 0.01. (B) WCE from RA3331, RA3331^SLX4 and RA3331^SLX4ΔSAP were analyzed by WB using indicated antibodies. (C) IFNα, IFNβ and IFNγ mRNA levels were measured in RA3331^SLX4 treated with conditioned medium from RA3331^SLX4 or RA3331 pre-incubated or not with neutralizing antibody to human IFNα, IFNβ and IFNγ. Graph represents mean (± SD) mRNA levels of one representative experiment relative to cells treated with conditioned medium from RA3331^SLX4. See also Fig. S1.

Fig. 2

Cytoplasmic accumulation of ssDNA is responsible for pro-inflammatory signals in SLX4 deficiency. (A) Immunofluorescence analysis was performed on RA3331, RA3331^SLX4 and RA3331^SLX4ΔSAP using ssDNA-specific antibody and DAPI nuclear staining. Images are representative of at least 4 independent experiments. (B) Quantification of staining observed in A. (C) Left panel: experimental scheme. Briefly, cytoplasmic extraction was performed on RA3331, RA3331^SLX4 and RA3331^SLX4ΔSAP followed by RNase cocktail (RNase A and RNase T1) and proteinase K treatments prior to DNA extraction and radiolabeling. DNA extracted from RA3331 cells was subjected or not to a subsequent S1 Nuclease treatment prior to separation on acrylamide gel and autoradiography. Right panel: autoradiography of a representative experiment. (D) WCE from RA3331 expressing Luciferase-, MYD88-, or STING-targeting inducible shRNAs were analyzed by WB using indicated antibodies. (E) Graphs present RT-qPCR quantification of knock-down efficiency, measured using specific primers, as mean (± SD) mRNA expression relative to cells expressing Luciferase-targeting shRNA from 3 independent experiments. (F) MxA, RIG-I, TLR4 and TLR2 mRNA levels were measured in cells described in D. Graphs present means (± SD) of 3 independent experiments as fold increase mRNA expression relative to cells expressing a Luciferase targeting shRNA. (G) WCE from RA3331 expressing Luciferase, STING, cGAS or IFI16-targeting inducible shRNAs were analyzed by WB using indicated antibodies. (H) MxA, STING, cGAS and IFI16 mRNA levels were measured in cells described in G. Graphs present means (± SD) of 3 independent experiments expressed as fold change in mRNA expression relative to cells expressing a Luciferase targeting shRNA. ****: p < 0.001; ***: p < 0.005; **: p < 0.01. See also Fig. S2.

Fig. 3

The SLX4com regulates cytoplasmic accumulation of LINE-1 DNA. (A) LINE-1 DNA was measured by qPCR in the cytoplasmic fraction of RA3331 or RA3331^SLX4. #1: primers targeting the 5′ of ORF1. #2: primers targeting the 3′ of ORF2. (B) Cytoplasmic DNA extracted from RA3331 or RA3331^SLX4 were analyzed by Southern Blot using LINE-1 specific probe. The right panel shows the molecular weight ladder. (C) Same as in A, except that primers allowing the amplification of active LINE-1 elements were used. #3 and #4: primer pairs targeting the 3′ UTR sequence of LINE-1 Ta-1 sub-family. (D) Constructs used in E-G and I. (E) FLAG/HA-WT-SLX4 or FLAG/HA-SLX4ΔSAP were FLAG-immunoprecipitated from 293T cells in the presence of a LINE-1-expressing plasmid. Bound material was peptide-eluted. Input and eluates were analyzed by WB using indicated antibodies. (F) FLAG-WT-MUS81 or FLAG-MUS81ΔWH were FLAG-immunoprecipitated from 293T cells in the presence of a LINE-1-expressing plasmid. Bound material was peptide-eluted. Input and eluates were analyzed as in E. (G) FLAG-WT-MUS81 was purified as in E in the presence or absence of 0.1 mg/ml EtBr and 0.1 U/μl DNaseI. Input and eluates were analyzed as in E. (H) LINE-1 construct used in I. Primers are indicated in red. (I) FLAG/HA-WT-SLX4, FLAG/HA-SLX4ΔSAP, FLAG-WT-MUS81 or FLAG-MUS81ΔWH were purified as in E prior to DNA extraction. Samples were analyzed by qPCR using primers indicated in H. See also Fig. S3.

Fig. 4

The SLX4 complex negatively regulates LINE-1 retrotransposition. (A) LINE-1 retrotransposition assay was performed in HeLa cells overexpressing either WT-SLX4 or SLX4ΔSAP together with a LINE1-NEO reporter plasmid. Following neomycin selection, resistant clones were crystal violet stained. Left panel presents one representative experiment. Graph represents crystal violet staining quantified over 3 independent experiments relative to cells expressing an empty vector. (B) LINE-1 retrotransposition assay was performed as in A, except that HeLa cells were treated with scrambled siRNA or siRNAs targeting SLX4 or MUS81. Graphs represent quantification as in A. (C) LINE-1 constructs used in D. LINE-1-ORF1mut contains the JM111 mutation in the ORF1 sequence that abolishes retrotransposition. (D) LINE-1 retrotransposition assay performed in RA3331 or RA3331^SLX4 transfected with plasmids described in C. Means (± SD) Luciferase activity of triplicate measurement from 4 independent experiments is presented as a percentage of maximum signal intensity. ****: p < 0.001. See also Fig. S4.

Fig. 5

Absence of FANCD2 leads to upregulated STING-dependent IFN production. (A) Retrotransposition was measured and results presented as in Fig. 4A except that cells were treated with scrambled siRNA or siRNA targeting FANCD2. (B) IFNα, IFNβ, IFNγ and MxA mRNA levels were measured in FANCD2 deficient cells and on their WT-FANCD2 complemented counterparts. Graphs present means (± SD) from triplicate measurement of 6 independent experiments expressed as fold mRNA expression relative to FANCD2 proficient cells. (C) Immunofluorescence was performed on FANCD2 deficient or proficient cells as in Fig. 2A. (D) WCE from PD20 expressing Luciferase, STING, cGAS or IFI16-targeting inducible shRNAs were analyzed by WB using indicated antibodies. (E) MxA, STING, cGAS and IFI16 mRNA levels were measured in cells described in D. Graphs present means (± SD) of 4 independent experiments expressed as fold change in mRNA expression relative to cells expressing Luciferase targeting shRNA. ****: p < 0.001; ***: p < 0.005; **: p < 0.01. See also Fig. S5.

Fig. 6

Upregulated retrotransposition leads to pro-inflammatory signaling. (A) IFNα, IFNβ, IFNγ and MxA mRNA levels were measured in RA3331 cells following 48 h treatment with 125 μM TenoF. Graphs present means (± SD) from triplicate measurement of 6 independent experiments expressed as fold change in mRNA expression relative to RA3331. ****: p < 0.001; ***: p < 0.005; **: p < 0.01. (B) IFNα, IFNβ, IFNγ and MxA mRNA levels were measured in FANCD2-deficient cells following 48 h treatment with 5 μM TenoF. Graphs present means (± SD) from triplicate measurement of 6 independent experiments expressed as fold change in mRNA expression relative to untreated cells. ****: p < 0.001; ***: p < 0.005; **: p < 0.01. (C) Cytoplasmic DNA was prepared from RA3331 cells following 8 h treatment with 125 μM TenoF, radiolabeled and analyzed by autoradiography. (D) DNA was prepared as described in C and analyzed by qPCR using primers described in Fig. 3A. Graph presents data from a representative experiment. (E) IFNα, IFNβ, IFNγ and MxA mRNA levels were measured in RA3331^SLX4 treated or not with 125 μM TenoF 24 h prior to treatment with 10 μM Cisplatin. Graphs present means (± SD) from 3 independent experiments as fold change in mRNA expression, relative to untreated cells. ****: p < 0.001; ***: p < 0.005; **: p < 0.01; *: p < 0.05. (F) WCE from cells treated as in E were analyzed by WB. See also Fig. S6.

Fig. 7

Inhibition of LINE-1 retrotransposition by the SLX4com prevents pro-inflammatory signaling. (A) The SLX4com inhibits LINE-1 retrotransposition by preventing accumulation of reverse transcribed LINE-1 DNA. LINE-1-derived DNA are likely subsequently degraded. (B) In the absence of the SLX4com, by-products of LINE-1 reverse-transcription accumulate in the cytoplasm. These are recognized by the cGAS-STING pathway to activate pro-inflammatory signaling. Grey lines represents: LINE-1 RNA; green lines: LINE-1 reverse-transcribed DNA.