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Destabilizing heterochromatin by APOE mediates senescence

Nature Aging volume 2, pages 303–316 (2022)Cite this article

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Abstract

Apolipoprotein E (APOE) is a component of lipoprotein particles that function in the homeostasis of cholesterol and other lipids. Although APOE is genetically associated with human longevity and Alzheimer’s disease, its mechanistic role in aging is largely unknown. Here, we used human genetic, stress-induced and physiological cellular aging models to explore APOE-driven processes in stem cell homeostasis and aging. We report that in aged human mesenchymal progenitor cells (MPCs), APOE accumulation is a driver for cellular senescence. By contrast, CRISPR–Cas9-mediated deletion of APOE endows human MPCs with resistance to cellular senescence. Mechanistically, we discovered that APOE functions as a destabilizer for heterochromatin. Specifically, increased APOE leads to the degradation of nuclear lamina proteins and a heterochromatin-associated protein KRAB-associated protein 1 via the autophagy–lysosomal pathway, thereby disrupting heterochromatin and causing senescence. Altogether, our findings uncover a role of APOE as an epigenetic mediator of senescence and provide potential targets to ameliorate aging-related diseases.

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Fig. 1: APOE expression increases in senescent human MPCs.
Fig. 2: Depletion of APOE attenuates cellular senescence in human MPCs.
Fig. 3: APOE promotes the degradation of nuclear envelope and heterochromatin-associated proteins.
Fig. 4: APOE interrupts nuclear envelope homeostasis and heterochromatin organization.
Fig. 5: Depletion of APOE stabilizes heterochromatin.
Fig. 6: Knockdown of APOE exhibits a geroprotective effect in senescent human MPCs.

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Data availability

The high-throughput sequencing data including RNA-seq, DamID-seq, ATAC-seq and whole-genome sequencing generated in this study have been deposited with the Genome Sequence Archive in the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation) of the CAS under accession no. HRA000668 and are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/browse/HRA000668. MS data have been deposited with the ProteomeXchange Consortium via the PRoteomics IDEntifications partner repository with the dataset identifier PXD024414.

Code availability

All custom analysis code and scripts for reproducing the key conclusions in this study can be found at https://github.com/QianzhaoJ/APOE_2021.

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Acknowledgements

We thank L. Bai, R. Bai, S. Ma, J. Lu, J.Chen and Y. Yang for their administrative assistance, X. Zhang, W. Li, J. Jia and J. Jiao for their help with the animal experiments, L. Wang, J. Yang, L. Fu, W. Wang, C. Liang, Z. Liu for their technical support, J. Wang for his help with the LC–MS/MS assay, J. Jia and S. Sun for their help with the FACS experiment and C. Peng for her help with the TEM sample preparation. The processing of high-throughput sequencing data was conducted on the ‘Era’ Petascale supercomputer of the Computer Network Information Center of the CAS. This work was supported by the National Key Research and Development Program of China (no. 2018YFC2000100), the Strategic Priority Research Program of CAS (no. XDA16010000), Beijing Natural Science Foundation (nos. Z190019, JQ20031), the National Key Research and Development Program of China (nos. 2021YFF1201000, 2020YFA0112200, 2020YFA0113400, 2020YFA0804000, 2020YFA0803401 2019YFA0802202, 2019YFA0110100, 2018YFA0107203, 2018YFC2000400 2017YFA0103304 and 2017YFA0102802), the National Natural Science Foundation of China (nos. 81901433, 81921006, 81625009, 91749202, 81861168034, 91949209, 82125011, 92049304, 92049116, 81822018, 81870228, 81922027, 82071588, 82122024, 32100937, 32121001, 31970597 and 81801399), the Program of the Beijing Municipal Science and Technology Commission (no. Z191100001519005), the Key Research Program of the Chinese Academy of Sciences (no. KFZD-SW-221), the K. C. Wong Education Foundation (nos. GJTD-2019-06 and GJTD-2019-08), the 14th Five-year Network Security and Informatization Plan of CAS (no. WX145XQ07-18), the Science and Technology Service Network Initiative of CAS (no. KFJ-STS-QYZD-2021-08-001), CAS Special Research Assistant Program, Youth Innovation Promotion Association of CAS (nos. E1CAZW0401 and 2020085), CAS Project for Young Scientists in Basic Research (YSBR-012), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (no. 2020-JKCS-011), the State Key Laboratory of Stem Cell and Reproductive Biology, the State Key Laboratory of Membrane Biology and the Milky Way Research Foundation.

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Author notes

  1. These authors contributed equally: Hongkai Zhao, Qianzhao Ji, Zeming Wu, Si Wang.

Authors and Affiliations

  1. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Hongkai Zhao, Zeming Wu, Zehua Wang, Qun Chu, Zhejun Ji & Jing Qu

  2. University of Science and Technology of China, Hefei, China

    Hongkai Zhao & Jing Qu

  3. University of the Chinese Academy of Sciences, Beijing, China

    Qianzhao Ji, Jie Ren, Zehua Wang, Jianli Hu, Qiaoran Wang, Moshi Song, Weiqi Zhang, Jing Qu & Guang-Hui Liu

  4. State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Qianzhao Ji, Zeming Wu, Kaowen Yan, Huifang Hu, Yusheng Cai, Jingyi Li, Moshi Song & Guang-Hui Liu

  5. Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China

    Zeming Wu, Jie Ren, Kaowen Yan, Qun Chu, Huifang Hu, Yusheng Cai, Zhejun Ji, Jingyi Li, Moshi Song, Weiqi Zhang, Jing Qu & Guang-Hui Liu

  6. Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China

    Zeming Wu, Kaowen Yan, Qun Chu, Huifang Hu, Yusheng Cai, Zhejun Ji, Jingyi Li, Moshi Song, Jing Qu & Guang-Hui Liu

  7. Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital, Capital Medical University, Beijing, China

    Si Wang, Daoyuan Huang & Guang-Hui Liu

  8. Chongqing Renji Hospital, University of the Chinese Academy of Sciences, Chongqing, China

    Si Wang

  9. Chinese Academy of Sciences Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China

    Jie Ren, Jianli Hu, Qiaoran Wang & Weiqi Zhang

  10. China National Center for Bioinformation, Beijing, China

    Jie Ren, Jianli Hu, Qiaoran Wang & Weiqi Zhang

  11. Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    Juan Carlos Izpisua Belmonte

  12. Aging Translational Medicine Center, International Center for Aging and Cancer, Xuanwu Hospital, Capital Medical University, Beijing, China

    Guang-Hui Liu

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  1. Hongkai Zhao
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  2. Qianzhao Ji
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  20. Guang-Hui Liu
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Contributions

G.H.L., J.Q., W.Z. and M.S. conceived the work and supervised the experiments. H.Z., Z. Wu and K.Y. performed the phenotypic and mechanistic analyses. Q.J. and S.W. performed bioinformatics analyses. Z. Wu, Z. Wang and J.H. performed the RNA-seq, DamID-seq and ATAC-seq library construction. Q.C. performed RNA and DNA analyses. H.H. and Y.C. performed immunostaining analysis and animal experiments. Q.W. performed the plasmid construction. D.H. and Z.J. performed the lentivirus packaging and protein stability analysis. G.H.L., J.Q., W.Z., M.S., H.Z., Q.J., Z. Wu and S.W. performed the data analysis. G.H.L., J.Q., W.Z., M.S., J.C.I.B., H.Z., Q.J., Z. Wu, S.W., J.R. and J.L. wrote the manuscript.

Corresponding authors

Correspondence to Moshi Song, Weiqi Zhang, Jing Qu or Guang-Hui Liu.

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The authors declare no competing interests.

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Nature Aging thanks Karl Riabowol, Zhixun Dou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Generation and characterization of APOE+/+ and APOE-/- hESCs.

a, RT-qPCR showing the mRNA levels of APOE and CDKN1A (P21) in RS-hMPCs (P3), HGPS-hMPCs (P3), and WS-hMPCs (P3). GAPDH was used as the internal control. b, Western blot analysis of APOE in hMPCs (P4) transduced with lentiviruses expressing luciferase (Luc) or APOE. c, Schematic diagram of CRISPR-dCas9-mediated transcriptional activation of endogenous APOE. d, Western blot analysis of APOE in hMPCs transfected with non-targeting (negative control, sg-NC) or APOE-targeting (sg-APOE) sgRNAs using the CRISPR-dCas9 transcriptional activation system. e, Schematic diagram of APOE knockout strategy through CRISPR-Cas9-mediated non-homologous end joining (NHEJ). Sequencing results showed a 1-bp (T/A) insertion introduced by genome editing. f, Western blot analysis of the APOE protein level in APOE+/+ hESCs and APOE-/- hESCs. g, Immunofluorescence analysis of pluripotency markers OCT4, NANOG, and SOX2 in APOE+/+ hESC and APOE-/- hESC. Scale bars, 25 μm. Phase-contrast images were shown on the right. Scale bars, 250 μm. h, Immunofluorescence analysis of TuJ1, SMA, and FOXA2 to evaluate the differentiation potential of APOE+/+ hESC and APOE-/- hESC toward lineages of all three germ layers, that is, ectoderm, mesoderm, and endoderm, respectively. Scale bars, 25 μm. i, Cell cycle analysis of APOE+/+ hESCs and APOE-/- hESCs. j, Immunofluorescence analysis of Ki67 in APOE+/+ hESCs and APOE-/- hESCs. Scale bars, 25 μm. k, Detection of the DNA methylation status of the OCT4 promoter in APOE+/+ hESCs and APOE-/- hESCs. l, Karyotype analysis of APOE+/+ hESCs and APOE-/- hESCs. m, The copy number variation (CNV) analysis by whole genome sequencing of APOE+/+ hESCs and APOE-/- hESCs. a,i–j, A Two-tailed Student’s t-test was used for statistical analysis. Data are presented as the means ± s.e.m. a, n = 4 biological repeats. b,d,f,g, n = 3 biological repeats. b,d,f, For western blot analysis, β-Tubulin was used as the loading control.

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Extended Data Fig. 2 Characterization of APOE+/+ and APOE-/- hMPCs.

a, Absence of markers (CD34, CD43, and CD45) irrelevant to hMPCs by flow cytometry analysis in APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs. b, Characterization of the osteogenesis capacity of APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs by Von Kossa and Osteocalcin staining. c, Characterization of the adipogenesis capacity of APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs by Oil Red O and FABP4 staining. d, Characterization of the chondrogenesis capacity of APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs by Toluidine blue staining. e, CNV analysis of APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs. f, Heatmap showing the Euclidean distance of RNA-seq replicates in APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs. The color key from dark to light represents relatively low to high Euclidean distance, respectively. g, Gene set enrichment analysis showing that SASP-associated genes were downregulated in APOE-/- (P3) hMPCs compared to APOE+/+ (P3) hMPCs. h, Immunofluorescence analysis of γH2AX foci in APOE+/+ (P9) hMPCs and APOE-/- (P9) hMPCs. i, Western blot analysis of APOE in APOE-/- (P9) hMPCs transduced with lentiviruses expressing luciferase (Luc) or APOE. β-Tubulin was used as the loading control. b–d,h, A Two-tailed Student’s t-test was used for statistical analysis. b–d,h–i, Data are presented as the means ± s.e.m. n = 3 biological repeats. b-d, Scale bars, 50 μm. h, Scale bars, 25 μm.

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Extended Data Fig. 3 APOE interacts with nuclear envelope proteins and heterochromatin-associated proteins.

a, Networks showing the potential APOE-interacting proteins associated with ‘Chromosomal region’ and ‘Nuclear envelope’ Gene Ontology (GO) annotations. The color key represents relatively low to high coverages of APOE-interacting proteins, respectively. b, Co-IP analysis in APOE+/+ (P4) hMPCs and APOE-/- (P4) hMPCs indicated no interaction between APOE and Nucleolin or β-Tubulin, referred to Fig. 3c as negative controls. c, Immunofluorescence analysis of APOE-FLAG and Emerin in APOE-/- (P9) hMPCs transduced with lentiviruses expressing FLAG-tagged APOE (left). Fluorescence signal density plots show the distributions of Emerin (red) and APOE (green) signals in APOE-/- hMPCs, as measured between peripheries. Individual cell measurements are shown as thin lines and average curves are shown as thick lines (right). d, Western blot analysis of APOE in APOE+/+ hMPCs and APOE-/- hMPCs transduced with lentiviruses expressing FLAG-tagged APOE. e, Protein stability analysis of KAP1, LBR and Emerin in APOE+/+ (P4) hMPCs transduced with lentiviruses expressing Luc-FLAG or APOE-FLAG. Protein levels of KAP1, LBR and Emerin at indicated time points in the presence of a protein synthesis inhibitor cycloheximide (CHX) treatment were determined by western blotting. f, Immunofluorescence analysis of H3K9me3 in APOE-/- (P4) hMPCs transduced with lentiviruses expressing FLAG-tagged APOE, 3×NLS-tagged APOE and 3×NES-tagged APOE. g, Immunofluorescence analysis of Ki67 in APOE-/- (P4) hMPCs transduced with lentiviruses expressing FLAG-tagged APOE, 3×NLS-tagged APOE and 3×NES-tagged APOE. h, Co-IP verification for the interaction between LC3 and APOE-FLAG in HEK293T cells. i, Immunofluorescence analysis of Emerin and LAMP2 in APOE+/+ hMPCs transduced with lentiviruses expressing FLAG-tagged APOE and treated with 50 nM bafilomycin A1 (BFA1) for 12 h. j, Western blot analysis of heterochromatin proteins in APOE+/+ hMPCs and APOE-/- hMPCs after treatment with si-NC or si-ATG5. d,e, β-Tubulin was used as the loading control. j, GAPDH was used as the loading control. e–g,j, A Two-tailed Student’s t-test was used for statistical analysis. b,d,h–i, n = 3 biological repeats. c, n=13. e,g,j, n = 3 independent experiments. f, n=150 cells per group. c,i, Scale bars, 10 μm. f,g, Scale bars, 25 μm.

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Extended Data Fig. 4 DamID-seq and ATAC-seq analyses of APOE+/+ and APOE-/- hMPCs.

a, Heatmap showing the Euclidean distance of DamID-seq replicates in APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs. The color key from dark to light represents relatively low to high Euclidean distance, respectively. b, Heatmaps showing increased DamID signals at LINE1, Satellite, and Alu regions in APOE-/- (P3) hMPCs compared to APOE+/+ (P3) hMPCs. The color key from blue to red represents relatively low to high DamID signals, respectively. c, Heatmap showing the Euclidean distance of ATAC-seq replicates in APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs. The color key from dark to light represents relatively low to high Euclidean distance, respectively. d, Left, scatter plot showing the differential ATAC-seq peaks identified by Diffbind in APOE-/- (P3) hMPCs compared to APOE+/+ (P3) hMPCs. Red and blue dots represent the opened and closed ATAC-seq peaks in APOE-/- hMPCs, respectively. Right, heatmaps showing the ATAC signals within opened and closed ATAC-seq peaks in both APOE+/+ (P3) hMPCs and APOE-/- (P3) hMPCs. e, Genomic element distribution analysis of opened and closed ATAC peaks in APOE-/- (P3) hMPCs compared to APOE+/+ (P3) hMPCs. f, Heatmap showing decreased ATAC signals ranging from 5-kb upstream to 5-kb downstream of ATAC-seq peaks at LAD-localized LINE1, Satellite, and Alu regions in APOE-/- (P3) hMPCs compared to APOE+/+ (P3) hMPCs. The color key from light to dark represents relatively low to high ATAC signals, respectively.

Extended Data Fig. 5 Knockdown of KAP1 or LBR accelerates cell senescence in APOE-/- hMPCs.

a, Western blot analysis of KAP1 in APOE-/- (P3) hMPCs transduced with lentiviral sh-GL2 or sh-KAP1. b, Clonal expansion assay in APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-KAP1. c, Immunofluorescence analysis of Ki67 in APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-KAP1. The white arrows indicate Ki67-positive cells. d, SA-β-gal staining of APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-KAP1. e, Western blot analysis of LBR in APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-LBR. f, Clonal expansion assay in APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-LBR. g, Immunofluorescence analysis of Ki67 in APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-LBR. The white arrows indicate Ki67-positive cells. h, SA-β-gal staining of APOE-/- (P4) hMPCs transduced with lentiviral sh-GL2 or sh-LBR. b–d,f–h, A Two-tailed Student’s t-test was used for statistical analysis. Data are presented as the means ± s,e,m. a,c–d,e,g–h, n = 3 independent experiments. b,f, n = 4 biological repeats. c–d,g–h, Scale bars, 25 μm. a,e, β-Tubulin was used as the loading control. c,g, The white arrows indicate Ki67+ cells.

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Extended Data Fig. 6 Knockdown of APOE delays physiological and pathological aging in hMPCs.

a, Western blot analysis of APOE in replicativesenescent human MPCs (P9), HGPS human MPCs (P9), Werner syndrome human MPCs (P9) and primary hMPCs (P8) from an aged individual transduced with lentiviral sh-GL2 or sh-APOE. β-Tubulin was used as the loading control. b, Nuclear DNA staining with Hoechst 33342 in replicativesenescent human MPCs (P9), HGPS human MPCs (P9), Werner syndrome human MPCs (P9) and primary hMPCs (P8) from an aged individual transduced with lentiviral sh-GL2 or sh-APOE. c, Immunofluorescence analysis of Ki67 in replicativesenescent human MPCs (P9), HGPS human MPCs (P9), Werner syndrome human MPCs (P9), and primary hMPCs (P8) from an aged individual transduced with lentiviral sh-GL2 or sh-APOE. d, SA-β-gal staining of replicativesenescent human MPCs (P9), HGPS human MPCs (P9), Werner syndrome human MPCs (P9), and primary hMPCs (P8) from an aged individual transduced with lentiviral sh-GL2 or sh-APOE. b–d, A Two-tailed Student’s t-test was used for statistical analysis. a,c–d, Data are presented as the means ± s,e,m. n = 3 independent experiments. b, n = 300 cells per group. b, Scale bars, 7.5 μm. c,d, Scale bars, 25 μm. a, β-Tubulin was used as the loading control. c, The white arrows indicate Ki67+ cells.

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Extended Data Fig. 7 Knockdown of APOE delays hMPC senescence induced by different stressors.

a–d, Phenotypic analyses of sh-APOE-infected hMPCs (P2) after H2O2 treatment including immunofluorescence analysis of H3K9me3 (a), SA-β-gal staining (b), immunofluorescence analysis of Ki67 (c) and clonal expansion assay (d). e–h, Phenotypic analyses of sh-APOE-infected hMPCs (P2) after UV irradiation including immunofluorescence analysis of H3K9me3 (e), SA-β-gal staining (f), immunofluorescence analysis of Ki67 (g) and clonal expansion assay (h). i–l, Phenotypic analyses of sh-APOE-infected hMPCs (P2) upon H-Rasv12 overexpression including immunofluorescence analysis of H3K9me3 (i), SA-β-gal staining (j), immunofluorescence analysis of Ki67 (k) and clonal expansion assay (l). a–l, A Two-tailed Student’s t-test was used for statistical analysis. a,e,i, n = 300 cells per group. b–c,f–g,j–k, n = 3 independent experiments. d,h,l, n = 4 biological repeats. a–c,e–g,i–k, Scale bars, 25 μm. c,g,k, The white arrows indicate Ki67+ cells.

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Extended Data Fig. 8 Knockdown of APOE delays human skin fibroblast senescence.

a–c, Phenotypic analysis of human skin fibroblasts (P10) transduced with lentiviruses expressing luciferase (Luc) or APOE. Immunofluorescence analysis of H3K9me3 (a) and Ki67 (b), clonal expansion assay (c) in human skin fibroblasts transduced with lentiviruses expressing Luc or APOE. d–f, Phenotypic analysis of human skin replicative-senescent fibroblasts (P14) transduced with lentiviral sh-GL2 or sh-APOE. Immunofluorescence analysis of H3K9me3 (d) and Ki67 (e), clonal expansion assay (f) in human skin fibroblasts transduced with lentiviral sh-GL2 or sh-APOE. g–i, Phenotypic analyses of sh-APOE-transduced human skin fibroblasts (P2) after H2O2 treatment including immunofluorescence analysis of H3K9me3 (g), immunofluorescence analysis of Ki67 (h) and clonal expansion assay (i). j–l, Phenotypic analyses of sh-APOE-transduced human skin fibroblasts (P2) upon H-Rasv12 overexpression including immunofluorescence analysis of H3K9me3 (j), immunofluorescence analysis of Ki67 (k) and clonal expansion assay (l). a–l, A Two-tailed Student’s t-test was used for statistical analysis. a,d,g,j, Data are presented as the means ± s.e.m. n = 150 cells per group. b,e,h,k, n = 3 independent experiments. c,f,i,l, n = 4 biological repeats. a,b,d,e,g,h, Scale bars, 25 μm. b,e,h,k, The white arrows indicate Ki67+ cells.

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Zhao, H., Ji, Q., Wu, Z. et al. Destabilizing heterochromatin by APOE mediates senescence. Nat Aging 2, 303–316 (2022). https://doi.org/10.1038/s43587-022-00186-z

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