Fig 1: CGI− gene misexpression drives age-associated secretory phenotypes.(A) GOCC analysis of all CGI+/− genes. Annotations of CGI+/− genes to depth-1 GOCC (the highest level in the GOCC hierarchy tree) are shown. P values were calculated using hypergeometric distributions. (B and D) Expression of genes encoding extracellular proteins in DO mouse kidneys (B) or upon chromatin architecture disruption (D). Shown are extracellular protein-coding genes that are significantly up/down-regulated in affected kidneys (versus unaffected, FDR < 0.05) (B) or upon nuclear architecture disruption (versus controls, at least threefold in three or more contexts) (D). For a full list of the genes and their curated functions (GOBP, GO Biological Process), see table S5 (A and C). (C and E) mRNA-FISH (Lcn2, a proinflammatory secretory CGI− gene) and immunofluorescence (H3K9me2/3) assays in kidneys from DO (C) or LBR-null (E) mice. White dotted lines indicate the boundaries of renal tubules. Note that nuclei with intact heterochromatin (white triangles; Het+) rarely misexpress secretory CGI− gene (Lcn2). (F) Meta-analysis of published plasma-proteome profiling data. (G) Portions of Lcn2-expressing cells among cells with and without senescence marker (p16INK4a mRNA) expression. See also fig. S8 for the p16INK4a mRNA and galactosidase β1 (GLB1) immunofluorescence staining. *P < 0.05, **P < 0.01, and ***P < 0.001. All error bars indicate SD of four replicates.
Fig 2: CGI- gene misexpression causes loss of cellular identity during aging.(A and C) Expression levels of tissue-specific genes in DO mouse kidneys (A) or upon chromatin architecture disruption (C). Shown are tissue-specific genes that are significantly up/down-regulated in affected kidneys [versus unaffected, false discovery rate (FDR) < 0.05; top part of (A)], kidney-specific genes [bottom part of (A)] or upon nuclear architecture disruption (versus controls, at least threefold in three or more contexts) (C). For a full list of the genes, see table S9 (A and H). Un, unaffected; Af, affected kidney. (B and D) mRNA-FISH (Gpnmb) and immunofluorescence (H3K9me2/3) assays in kidneys from DO (B) or LBR-null (D) mice. Note that nuclei with intact heterochromatin (white triangles; Het+) rarely misexpress nerve-specific CGI- gene (Gpnmb). White dotted lines indicate the boundaries of renal tubules. Error bars indicate SD of four replicates. (E) Age-associated increase in transcriptional noise (table S10). DRG, dorsal root ganglion; HSC, hematopoietic stem cell. *P < 0.05; **P < 0.01; ***P < 0.001; and not significant (n.s.), P = 0.05. DAPI, 4',6-diamidino-2-phenylindole.
Fig 3: Disruption of nuclear architecture during aging drives uncontrolled expression of CGI− genes.(A and C) Representative data showing expression (left), distribution shift, and net expression change (right) of CGI+/− genes in LBR-null mouse kidneys (A) or in the context of chromatin architecture disruption (C). For full data, see fig. S2D and table S4. (B) Western blot of heterochromatin marks/component in LBR-null mouse kidneys. (D) Overlaps among expression-changed genes upon nuclear lamina/heterochromatin disruption or during aging. Significance was calculated by permutation tests (n = 1000). Kd, kidneys; Mb, myoblasts. (E) Portion of genes within three chromosomal domains that are up-regulated upon nuclear lamina/heterochromatin disruption or during aging [i.e., the eight contexts shown in (D)]. The polar angle of each group indicates its proportion of all CGI+ or CGI− genes, while the radius indicates the up-regulated percentage within each group. Recurrently up-regulated genes in at least two conditions of the eight contexts shown in Fig. 2D are shown in darker gray/red colors. (F) A representative genomic landscape of CGI+/− genes misexpressed during aging. †All Hi-C PC1 values of 28 cell/tissue types in this view and additional examples are illustrated in fig. S3.
Fig 4: HSV-1 redirects DDX3X to the nuclear rim at late time points.a HeLa cells grown on coverslips were either mock treated or infected with wild-type strain 17+ virus at an MOI of 5. The infection was stopped at 3, 6 or 9 h post-infection (hpi). Cells were fixed and processed for immunofluorescence (DDX3X: red; ICP4: green; Hoechst 33342: blue). Samples were imaged using STED microscopy. Scale bars represent 5 µm. The images represent three independent experiments. b Quantification of DDX3X relocalisation to the nucleus was performed by seeding HeLa cells on coverslips and mock treated or infected with wild-type strain 17+ virus at an MOI of 5. At 9 hpi, cells were fixed and processed for dual colour STED microscopy using anti-DDX3X (red) and anti-LBR (green) antibodies. In three independent experiments, twelve random whole cells (z-stacks) were imaged for both conditions. Next, images were processed with the Imaris software to calculate the amount of DDX3X found on a masked LBR channel. Scale bar represents 5 µm. ±Standard deviation of the means (bilateral Student T-tests; p = 0.0367).
Fig 5: DDX3X physically interacts with pUS3.HeLa cells were either mock treated or infected with wild-type strain 17+ virus for 12 h. Cells were then fractionated into cytoplasm, nuclear matrix and nuclear membranes as detailed in Methods and a probed by WB for the GAPDH and LBR cytoplasmic and nuclear membrane markers respectively and b used to immunoprecipitate DDX3X using the R648 polyclonal serum and monitored for the presence of pUS3. NFA: no first antibody. In parallel, total lysates were loaded on the gels. Results represent three independent experiments.
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