EMBO/SourceData huggingface dataset:
Figure 5(A) WT and Srcap-/- ISCs were conducted for transcriptome microarray, followed by Gene Set Enrichment Analysis (GSEA) analysis for PPARδ target genes.(B) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 and *** p<0.001 by two-tailed Student's t-test.(C) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining. Scale bar, 50 μm.(D) ISCs were collected for organoid formation followed by quantitative RT-PCR. For Prdm16 overexpression, ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 by two-tailed Student's t-test.(E) mRNA levels of ISC signature genes were examined by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates.(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.
Figure 6(A) Representative images of intestines from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) were measured in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.(B) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.(C) Intestine tissues from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group) were collected for immunohistochemistry staining. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.(D) Immunofluorescence staining of intestine tissues from Srcapfl/fl; Lgr5GFP-CreERT2 and Restfl/fl; Lgr5GFP-CreERT2 mice. Scale bar, 50 μm.(E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel).(F) Rest+/+ and Rest-/- mice were treated with 10 Gy's radiation and sacrificed at the indicated time points. Intestinal tissues were collected for H&E staining. Scale bars, 100 μm. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5).(G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.
Figure 1(B-D) Immunofluorescent images of mouse liver (B), testis (C) and heart (D). H3K27ac, PolII-S5P, and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti-mouse ChIL-probe (red: H3K27ac and PolIIS5P) and anti-rabbit IgG (green: laminin). DNA was counterstained with Hoechst 33342. Scale Bar: 20 µm (left images), 10 µm (right images).(E) Genome browser images of ChIL-seq for H3K27ac and PolII-S5P and bulk tissue RNA-seq data at the Alb locus in liver tissues.
Figure 2(B) The IGV tracks of H3K27ac ChIL-seq at identified tissue-specific enhancers of Rxra, Gnat3, Eps8, and a house-keeping gene of Actb loci are shown with the replicates.(C) Specific motif enrichment analysis was conducted using chromVAR(Schep et al, 2017). Hierarchical clustering of deviation-Z scores of three replicates of each tissue is shown.(D) Super-enhancer identification. Tissue-specific enhancers are identified, so that they are listed more than twice (twice: blue, all: red) in the top 5% in all enhancer candidates and are not in the SEs of other tissues. Grey shades indicate the top 5% of tag count in enhancer candidates.(F) Hnf4ɑ binds to the SE at Alb gene loci.
Figure 3(B) Signal intensities of ChIL-seq correlated with the expression levels of genes. The lines indicate the average CPM of each expression group at TSS. The expression groups were assigned with respect to the expression levels (TPM) of genes.(E) The tissue-specific genes identified by PolII S5P ChIL-seq. The IGV tracks of all replicates of PolII S5P ChIL-seq are shown at each specific gene (Trf, Myh6, and Meig for the liver, heart, and testis, respectively). Actb is also shown as the ubiquitously expressed gene in the three tissues.
Figure 4(B) PolII S5P ChIL-seq signal of the marker genes of mature skeletal muscle (Acta1) and macrophages (Cd68).(F) Bulk-tissue expression levels (TPM) of the representative genes.(H) Activities of major cell types in muscle regeneration. The colors of the heatmap show the log2FC to day 0 (uninjured) of TR and TSS levels. Representative genes with significant changes in TR are shown.
Figure 5(A) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues. For each lane, individual samples were selected from each progressive stage of breast cancer.(C) Expression, TSS level, and TR of genes. EEF1A1 is shown as a control housekeeping gene. The horizontal dotted line indicates the median level of all genes. Error bars indicate 95% confidence intervals for each estimate indicated by dots (N=1 for each).
Figure 1A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain.C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed).H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression.
Figure 2D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). Data information: Bars and error bar indicates mean ± SEM.
Figure 3A. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A, Kmt2A and Kmt2b knock out mice. Fisher's hypergeometric test, Benjamini-Hochberg (BH) correction.
Figure 4E. Cumulative hippocampus-dependent strategy scores during the Barnes maze training (One-way ANOVA). n = 10 per group. F. Number of visits to the escape hole during probe test (n = 10/group, One-Way ANOVA). Data information: Bars and error bars indicate mean ± SEM.
Figure 5E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).
Figure 1dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR
Figure 2dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated 9 days later with 300 U/ml IFNα (B) or 200 U/ml IFNγ (C) for indicated times (h: hours). Expression of indicated genes was analyzed by qRT-PCR relative to TATA-box binding protein (TBP) mRNA (n=6 biological replicates of two independent experiments).
Figure 3Liver tissue samples obtained from HBV-negative patients undergoing metastasis resection (Control), or patients with acute, self-limiting HBV infection or chronic hepatitis B, or chronic coinfection with HBV and hepatitis D virus (HDV) were stained for ISG20 by immunohistochemistry. For each clinical entity, tissue sections from three different patients are shown; scale bar: 50 µm. ISG20 positive area of each sample (n=3 biological replicates) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned.
Figure 4dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 10 to 14 p.i. and ISG20 was stained by immunofluorescence. Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. and ISG20 was stained by immunofluorescence.dHepaRG cells were infected with HBV at an MOI of 500 virions/cell, treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 7 to 9 p.i. and stained by immunofluorescence for ISG20 and PML (C) or ISG20 and nucleophosmin (D).
Figure 5dHepaRG cells were infected at an MOI of 100 virions/cell and transduced at day 7 p.i. with an adenoviral vector for expression of shRNA targeting ISG20 (AdV-shISG20). After 2 days, cells were treated with 300 U/ml IFNα or 200 U/ml IFNγ. ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment, respectively.
Figure 6A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated.
Figure 7dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and at day 7 p.i. transduced with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. Cell lysates were analyzed for cccDNA after T5 digest (B) and total intracellular HBV DNA (C) by qPCR relative to Prnp (n=5-6 biological replicates from two independent experiments).dHepaRG-TR-A3A cells were infected with wildtype HBV (HBVwt) or HBV∆x at an MOI of 300 virions/cell and transduced on day 7 p.i. with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. HBeAg (E) was measured by ELISA and cccDNA after T5 digest (F) by qPCR relative to Prnp (n=6 biological replicates from two independent experiments).
Figure 1(E) Normalized expression levels of the native core promoters. Their activities spanned over a broad range of three orders of magnitude (promoter constructs contained block 1.11 and block 7.11 combination as nucleosomal sequences). Each color represents a different class of core promoter architecture. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (including new cell transfection procedures and measurements of promoter activities).(I) Scatter plot of expression levels obtained in D. mel. S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D. mel. embryos, revealing a high correlation (Pearson coefficient 0.91) between the two datasets. Error bars represent standard deviations of 3-4 biological replicate measurements.
Figure 2(C) Boxplots depicting block 1 effects for tested core promoters. Effects of different block 7s were merged in each column (within the same block 1): the median SD is 0.66 for developmental promoters compared to 1.23 for constitutive promoters (lower right corner); Wilcoxon rank-sum test ***p = 3.1×10-4, significant. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (D) Boxplots depicting block 7 effects for tested core promoters. Effects of different block 1s were merged in each column (within the same block 7): the median SD is 0.54 for developmental promoters compared to 0.64 for constitutive promoters (lower right corner); Wilcoxon rank-sum test p = 0.3, not significant. Block 7 with column "w/o B7" represents the results obtained from promoters without block 7 sequence. Developmental and constitutive promoters are highlighted in green and red, respectively. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements.
Figure 3(B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements.
Figure 4(A) Consensus replacement. Heatmap depicting the mean expression fold changes compared to wild-type expressions after replacing with motif consensus sequences derived by XXmotif. Constitutive and developmental promoters are highlighted in red and green, respectively.
Figure 7(B) Expression fold changes (ecdysone inducibility) versus measurements without ecdysone and grouped by native core promoter sequences. The colors refer to different core promoter architectures. Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2.(C) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters. Disrupted INR (highlighted with the black dotted line rectangle) had a slightly negative effect on changing the core promoter responsiveness to ecdysone. (~ 2.3-fold reduction on average, Wilcoxon rank-sum test p = 2.1×10-5). Constitutive and developmental promoters highlighted in red and green, respectively.
Figure 1(B) Violin plots displaying RNA-seq readings by FPKM (normalized Fragments Per Kilobase Million) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls (n = 49,42; unpaired Kolmogorov-Smirnov test; continuous lines: median; dashed lines: quartiles).(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.(F-G) Mean and SEM of relative levels of S-nitrosylated IRE1 (f) and total IRE1 (g) in brains from young and aged mice. Histograms show relative levels of SNO-IRE1/input IRE1α measured by biotin-switch assay and total IRE1/GAPDH quantified by densitometry on standard immunoblots for young (3-month-old) versus aged (24 month-old) mouse brains (n = 3, unpaired Student's t test). Data information:
Figure 2Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (C) Representative photomicrographs of β-galactosidase staining of hippocampal slices derived from young (3 months), middle-aged (12 months) and aged (22-24 months) of IRE1WT or IRE1cKO animals. Magnification: 20x; scale bar: 200 μm. Arrows indicate β-galactosidase staining.Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (D) Mean and SEM of percentage of β-galactosidase staining in CA3 region (n = 3-5 animals/group).(F) Representative confocal images of dendritic spines (arrows) in the CA1 region of middle-aged IRE1WT animals injected with AAV-Mock or AAV-Cre. 60x magnification, (G) Mean and SEM of spine density per μm (n = 3, 3; 4, 4 animals). P computed by unpaired Student's t test within each age-matched group.
Figure 3(E) Representative images of β-galactosidase staining of hippocampal slices derived from aged (22-24 months) TgXBP1s and WT animals. (F) Mean and SEM of percentage of area stained by β-galactosidase in the hippocampus of aged (20-24 months) TgXBP1s compared to littermate controls.
Figure 6(D) Left panel: Representative images showing dendritic spines of aged (22-24 months) animals injected with AAV2-eGFP (mock) or with AAV2-XBP1s (arrows indicate dendritic spines). Young animal dendrite is shown for reference. 60x magnification. Scale bar: 5μm. Right panel: Mean and s.e.m. of spine density per μm for indicated experimental groups (n = 24, 53 and 35 dendrites from 3, 5 and 6 animals, respectively(E) Left panel: Representative images of β-galactosidase staining of hippocampal slices derived from aged mice injected with AAV2-Mock or AAV2-XBP1s (arrow indicate β-galactosidase staining). Magnification 20x; scale bar, 200 μm. Right panel: Histograms show mean and s.e.m. of percentage of β-galactosidase-stained area. n = 6, 6; unpaired Student's t test.(F) Left panel: Immunofluorescence of γH2aX (red) counterstained with nuclei (blue) comparing aged mice injected with AAV-Mock or AAV-XBP1s. Magnification: 40x; Scale bar: 50μm. Right panel: Histograms show mean and s.e.m. of percentage of γH2A- positive cells; unpaired Student's t test comparing the aged groups ( n = 6, 6 animals/group; unpaired Student's t test).
Figure 1B FACS analysis of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with DSF (5 µM) for 2 h (n = 3). Data information: , data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (B)C Representative FACS of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with the indicated concentrations of DSF for 2 h (n = 2). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (C)D Western blot analysis of the phosphorylation level of S6 and p70S6K in WT naïve CD8+ T cells stimulated by α-mouse CD3ε(145-2C11) (1 µgml-1) in the presence of α-mouse CD28 (1 µgml-1) for the indicated times after pretreatment with DSF (5 µM) for 2 h. The immunoblot shows normalized expression of p-S6(S235/236) to S6, p-p70S6K to p70S6K, p-Zap70 to Zap70, as well as that of p-LCK(Y394) and P-Lck(Y505) to Lck. Data information: , data are representative of 3 independent experiments, , Image J analysis in (D)E FACS analysis of p-CD3ζ(PY142) in mouse naïve CD8+ T cells treated with DSF or stimulated with α-mouse CD3ε(145-2C11) in the presence of α-mouse CD28 (1 µgml-1) for 2 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (E)F Western blot analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF for 2 h. The immunoblot analysis shows normalized expression of p-CD3ζ to CD3ζ as well as that of p-Zap70(Tyr319)/Syk(Tyr352), p-LCK(Y394), and p-LAT(Tyr191) to β-actin. Data information: data are representative of 3 independent experiments, Image J analysis in (F)G, H Luciferin assay of NFAT activity (G) and Nfatc1 mRNA expression (H) in J76-NFATRE-luc and TCR052 cells stimulated with α-human CD3 (OKT3) (1 µgml-1) in the presence of α-human CD28 (1 µgml-1) for 4 h or treated with DSF (5 µM) for 2 h. The levels of NFAT activity were normalized with TCR052 cells treated with DMSO (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; two-way analysis of variance (ANOVA) in (G, H)].