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. 2020 Jun 6;11(7):483–504. doi: 10.1007/s13238-020-00728-4

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Depletion of SIRT7 in hMSCs induces LINE1 activation and triggers innate immune responses via cGAS-STING pathway. (A) ChIP-qPCR assessment of SIRT7 enrichment at seven LINE1 regions in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). Data are presented as the means ± SEM. n = 3. **, P < 0.01. ***, P < 0.001 (t test). (B) ChIP-qPCR assessment of H3K9me3 enrichment of LINE1 regions in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6) using the fourth LINE1 primer. Data are presented as the means ± SEM. n = 4. ***, P < 0.001 (t test). (C) Violin plot showing the DamID signal [log₂ (Dam-EMD/ Dam)] in LINE1 regions located in LADs in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). The white circles represent the median values, and the white lines represent the values within the IQR from smallest to largest. ***, P < 0.001 (Two-sided Wilcoxon rank-sum test). (D) Violin plot showing the H3K9me3 signal in LINE1 regions located in “H3K9me3 mountains” in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). The white circles represent the median values, and the white lines represent the values within the IQR from smallest to largest. ***, P < 0.001 (Two-sided Wilcoxon rank-sum test). (E) Metaplot showing the average ATAC signal for ATAC peaks in LINE1 regions in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). ***, P < 0.001 (Two-sided Wilcoxon rank-sum test). (F) Quantitative RT-PCR analysis of LINE1 transcript levels in SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8). Data are presented as the means ± SEM. n = 4. *, P < 0.05. ***, P < 0.001 (t test). (G) Left, Western blot analysis of LINE1 ORF1 and ORF2 proteins in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6) with H3 used as loading control. Right, statistical analysis of relative ORF1 and ORF2 protein expression levels. Data are presented as the means ± SEM. n = 3. *, P < 0.05, **, P < 0.01 (t test). (H) Left, schematic of retrotransposition assay. The plasmid p99-GFP-LRE3-Cherry lacking a CMV promoter encodes a full-length LINE1 element tagged with an indicator cassette of mEGFP1 retrotransposition and mCherry cassette. Right, quantification of de novo retrotransposition events (EGFP-positive cells) in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6), normalized by live cell numbers and transfection efficiency. Data are presented as the means ± SEM. n = 3. *, P < 0.05. ***, P < 0.001 (t test). (I) RT-qPCR analysis of the relative LINE1 genomic DNA content in SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8). Data are presented as the means ± SEM. n = 4. *, P < 0.05. ***, P < 0.001 (t test). (J) Western blot analysis of the phosphorylation levels of P65, TBK1 and IRF3 in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6) with GAPDH used as loading control. (K) ELISA analysis of IL6 and MCP1 levels in culture medium of SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). IL6 levels were normalized to cell number. Data are presented as the means ± SEM. n = 5. MCP1 levels were normalized to cell number. n = 3. **, P < 0.001. ***, P < 0.001 (t test). (L) Heatmap showing quantitative RT-PCR analysis of IFN-I and SASP genes in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). Expression levels of these indicated genes in SIRT7^−/− hMSCs were normalized to those in SIRT7^+/+ hMSCs