HP1γ Prevents Activation of the cGAS/STING Pathway by Preserving Nuclear Envelope and Genomic Integrity in Colon Adenocarcinoma Cells

Abstract

Chronic inflammatory processes in the intestine result in serious conditions such as inflammatory bowel disease (IBD) and cancer. An increased detection of cytoplasmic DNA sensors has been reported in the IBD colon mucosa, suggesting their contribution in mucosal inflammation. Yet, the mechanisms altering DNA homeostasis and triggering the activation of DNA sensors remain poorly understood. In this study, we show that the epigenetic regulator HP1γ plays a role in preserving nuclear envelope and genomic integrity in enterocytic cells, thereby protecting against the presence of cytoplasmic DNA. Accordingly, HP1 loss of function led to the increased detection of cGAS/STING, a cytoplasmic DNA sensor that triggers inflammation. Thus, in addition to its role as a transcriptional silencer, HP1γ may also exert anti-inflammatory properties by preventing the activation of the endogenous cytoplasmic DNA response in the gut epithelium.

Keywords: HP1γ; STING; cGAS; cytosolic DNA; epigenetics; inflammation; inflammatory bowel disease; nuclear envelop.

Figure 1

Alterations in the nuclear envelope in TC7 Cbx3 KO cells. (A–F) Immunofluorescence of the nuclear envelope-associated protein LaminB1: large alterations can be seen in cells lacking HP1γ (D–F), whereas no abnormalities are seen in WT cells (A–C); (G,H) TEM analysis of nuclear envelope alterations in TC7 WT (G) or Cbx3 KO (H) cells: double membrane inclusions can be observed in cells lacking HP1γ (H, arrows). n = 5 clones analyzed per condition, 2 plates per clone. Scale bar: (A–F) 20 µm; (G,H) 1 µm.

Figure 2

Analysis of progerin production upon depletion of HP1γ in TC7 cells. (A–F) Immunofluorescence study provides evidence for a detection of progerin in the nucleus of TC7 Cbx3 KO cells (D–F) but not in TC7 WT (A–C). Images are representative of n = 5 clones (generated by 1 sgRNA) per condition, Scale bar: 25 µm.

Figure 3

Ultrastructural analysis of membranous inclusions observed in TC7 Cbx3 KO cells. (A) Irregular double membrane inclusion, observed next to the nuclear envelope. (B) Inclusion of the double spherical membrane, observed next to the nuclear envelope (arrows). The poorly electrodense nature of its contents can be appreciated. Arrows indicate the presence of ribosomes. (C) Inclusion of double spherical membrane, observed next to a large patch of heterochromatin (asterisk), suggesting the preference of these structures towards regions of gene repression. (D) Detail of small double spherical membrane inclusion, surrounded by ribosomes (arrows). n = 5 clones (generated by 1 sgRNA) per condition. Scale bar: 400 nm.

Figure 4

Ultrastructural analysis of membranous chromatin inclusions observed in the cytoplasm of TC7 Cbx3 KO cells. (A) Evidence for the presence of DNA-containing structures in the cytoplasm by staining with DAPI. (B,C) Examples of chromatin-containing double-membraned cytoplasmic inclusions (double membrane featured by arrows) observed in TC7 Cbx3 KO cells. While these inclusions are located close to the nuclear envelope, they are not in direct contact with it. (D) Detail of free chromatin structures (asterisk) observed in the cytoplasm of cells lacking HP1γ. These structures are found free in the cytoplasm, with no preference for any organelle or nuclear substructure. n = 5 clones (generated by 1 sgRNA) per condition. Scale bar: (A) 25 µm; (B–D) 400 nm.

Figure 5

Analysis of the induction of DNA damage foci upon loss of HP1γ in TC7 cells. (A–F) Immunofluorescence study for detecting DNA damage foci, defined by γH2AX detection in TC7 WT (A–C) and TC7 Cbx3 KO cells (D–F). (G) Quantitative analysis of γH2AX upregulation after loss of HP1γ in TC7 enterocytic cells. n = 5 clones (generated by 1 sgRNA) per condition, 2 plates per clone (in WT, different plates were analyzed together). Values are mean ± SD. **** p < 0.0001 (t-test and non-parametric, two-tailed p value). Scale bar: 25 µm.

Figure 6

Analysis of the c-GAS-inflammatory response upon depletion of HP1γ in TC7 cells: (A) Detection of cGAS by immunofluorescence microscopy: the cGAS signal was weakly visible in TC7 WT cells (A1–A3), and localized in the cell nucleus, whereas expression is triggered in TC7 Cbx3 KO cells and principally detected in the cytoplasm (A4–A6). (B) Quantitative analysis of the number of cGAS positive cells. (C) STING detection by immunofluorescence microscopy: STING protein remains at undetectable levels, based on our parameters in TC7 WT cells (C1–C3), while in TC7 Cbx3 KO cells (C4–C6), its presence in the cytoplasm was markedly induced. (D) Quantitative analysis of the number of STING positive cells. (E–I) Q-PCR analysis of the corresponding genes associated with the inflammatory response. n = 3 clones per condition (generated by 1 sgRNA), 3 plates per clone (different plates were analyzed together). Values are mean ± SD. * p < 0.05; *** p < 0.001; **** p < 0.0001 (t-test and non-parametric, two-tailed p value). Scale bar: 50 µm.