Journal: Nature Communications

Article Title: Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming

doi: 10.1038/s41467-018-08245-z

Figure Lengend Snippet: HCC methylome analysis identifies a recurrent hypomethylated enhancer targeting a liver-enriched transcriptional factor C/EBPβ . a A circos plot showing genomic regions that are significantly hypomethylated (4528 regions, in red) or hypermethylated (2654 regions, in green) in the tumor group as compared to matched non-tumors based on MBDCap-seq data. b Enrichment of DMRs in annotated functional elements, defined as ( Lm i / Lm )/( L i / L ), where Lm i is the total length of DMRs overlapping with the annotated functional elements of type i and Lm is the total length of all DMRs. L i is the length of the annotated functional elements of type i , and L is the total length of all annotated functional elements combined. c Genome-wide methylation patterns of differentially methylated FANTOM5 enhancers in HCC tumor (orange) and normal liver (blue) tissues based on WGBS. d Enhancer methylation levels of HCC tumor and normal liver tissues. e Relative methylation levels of 894 DMEs between HCC tumor and normal liver tissues. f ChIP-seq tracks of H3K27ac, H3K4me1, H3K4me3, and H3K27me3 at the 1-kb C/EBPβ enhancer locus (chromosome 20: 48,900,221–48,901,229) in HCC tumor tissues. g – i Methylation levels of 13 CpG sites within the C/EBPβ enhancer in 48 pairs of HCC tumor and non-tumor tissues as determined by pyrosequencing. j qRT-PCR analysis of HCC tumor and non-tumor tissues (33 pairs). C/EBPβ mRNA levels were calculated by the 2 −ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average value of the non-tumor group. k Correlation between C/EBPβ enhancer methylation and expression in 33 pairs of HCC tumor and non-tumor tissues. C/EBPβ mRNA levels are ΔCt values using 18s rRNA as internal control. l , m Kaplan–Meier survival analysis of 48 HCC patients according to their C/EBPβ hypomethylation statuses (relative methylation of tumor vs. non-tumor). Patients with strong hypomethylation (top 24) show poorer ( l ) overall and ( m ) disease-free survival rates than those with weak hypomethylation (bottom 24). Data are presented as mean ± SD. ** P < 0.01; **** P < 0.0001 as calculated by Wilcoxon signed-rank test ( d ), paired two-tailed Student’s t -test ( i , j ), Pearson correlation test ( k ) and Kaplan–Meier survival analysis ( l , m )

Article Snippet: The primary antibodies for western blotting are CEBPB (sc-150, Santa Cruz Biotechnology, 1:1000), β-actin (8H10D10, Cell Signaling Technology, 1:10,000), Vinculin (sc-25336, Santa Cruz Biotechnology, 1:1000), H3K27ac (39133, Active Motif, 1:1000), and H3 (4499, Cell Signaling Technology, 1:1000).

Techniques: Functional Assay, Genome Wide, Methylation, ChIP-sequencing, Quantitative RT-PCR, Control, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming

doi: 10.1038/s41467-018-08245-z

Figure Lengend Snippet: C/EBPβ auto-regulates its enhancer activity. a qChIP-PCR showed enrichment of C/EBPβ, BRD4, H3K27ac, and RNPII at C/EBPβ enhancer after 5-aza-dC treatment. b Western blot analysis of C/EBPβ level in liver cells upon siRNA-mediated knockdown. β-actin was used as loading control. c , d qRT-PCR analysis of C/EBPβ eRNA upon ( c ) C/EBPβ knockdown and ( d ) treatment with BRD4 inhibitor JQ1. C/EBPβ eRNA/mRNA levels were calculated by the 2 −ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average values of the respective control groups (siCtrl/DMSO). e Schematic diagrams of C/EBPβ promoter-, C/ EBPβ promoter- C/EBPβ enhancer-, and C/EBPβ promoter- C/EBPβ enhancer mutant-luciferase reporter constructs. The C/EBPβ promoter (−285 to + 808-bp relative to TSS) was cloned to the PGL3 vector, in the absence or presence of the C/EBPβ enhancer with the WT or deleted C/EBP consensus sequence. f The relative luciferase activity in liver cells upon transfection with different reporter constructs. g The relative luciferase activity of the C/EBPβ enhancer construct upon siRNA-mediated knockdown of C/EBPβ . Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 as calculated by unpaired two-tailed Student’s t -test ( a , c , d , f , g )

Article Snippet: The primary antibodies for western blotting are CEBPB (sc-150, Santa Cruz Biotechnology, 1:1000), β-actin (8H10D10, Cell Signaling Technology, 1:10,000), Vinculin (sc-25336, Santa Cruz Biotechnology, 1:1000), H3K27ac (39133, Active Motif, 1:1000), and H3 (4499, Cell Signaling Technology, 1:1000).

Techniques: Activity Assay, Western Blot, Knockdown, Control, Quantitative RT-PCR, Mutagenesis, Luciferase, Construct, Clone Assay, Plasmid Preparation, Sequencing, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming

doi: 10.1038/s41467-018-08245-z

Figure Lengend Snippet: C/ebpβ enhancer hypomethylation associates with C/EBPβ over-expression in HBx TG mice. a Patterns of C/EBPβ eRNA (CAGE reads, FANTOM5), C/EBPβ and H3K27ac binding in the human HepG2 (GEO: GSM935493) and mouse hepatocyte genomes (GEO: GSM1854433). b Pyrosequencing analysis of C/ebpβ enhancer in liver tissues of 4-month-old WT and HBx TG mice (WT, n = 7; TG, n = 14). c , d qRT-PCR analyses of c C/ebpβ eRNA and d C/ebpβ mRNA levels in liver tissues of 4- and 10-month-old WT and HBx TG mice (4-month-old WT, n = 7; 4-month-old TG, n = 12; 10-month-old WT, n = 7; 10-month-old, n = 12). C/ebpβ eRNA/mRNA levels were calculated by the 2 −ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average value of the 4-month-old WT group. e Western blot analysis of C/EBPβ protein level in liver tissues of 4- and 10-month-old WT and HBX TG mice (4-month-old WT, n = 7; 4-month-old TG, n = 7; 10-month-old WT, n = 6; 10-month-old, n = 8). Vinculin was used as loading control. The protein band intensities are quantified and shown on the right. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 as calculated by unpaired two-tailed Student’s t -test ( b – e )

Article Snippet: The primary antibodies for western blotting are CEBPB (sc-150, Santa Cruz Biotechnology, 1:1000), β-actin (8H10D10, Cell Signaling Technology, 1:10,000), Vinculin (sc-25336, Santa Cruz Biotechnology, 1:1000), H3K27ac (39133, Active Motif, 1:1000), and H3 (4499, Cell Signaling Technology, 1:1000).

Techniques: Over Expression, Binding Assay, Quantitative RT-PCR, Control, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming

doi: 10.1038/s41467-018-08245-z

Figure Lengend Snippet: C/EBPβ enhancer deletion impairs genome-wide enhancer activity and chromatin regulation of driver oncogene expressions. a Western blot analysis of C/EBPβ and H3K27ac levels in C/EBPβ enh +/− and C /EBPβ enh −/− compared to WT HepG2 cells. H3 and vinculin were used as loading controls. b Fold change (log 2 ) in C/EBPβ and BRD4 ChIP-seq signals at C/EBPβ binding sites in C /EBPβ enh −/− relative to WT HepG2 cells. c H3K27ac levels in WT and C /EBPβ enh −/− cells in TSS-proximal promoter ( + /− 2-kb) and TSS-distal enhancer ( > 2-kb) enrichment regions (the numbers in red, green, and gray of each plot denote the regions with H3K27ac-loss ( < 1/2 × ), -gain ( > 2 × ), and stable in C /EBPβ enh −/− cells). d , e ChIP-seq profiles of C/EBPβ, BRD4, and H3K27ac in WT and C /EBPβ enh −/− cells around all C/EBPβ/BRD4-codepleted d super-enhancer and e enhancer sites. The average ChIP-seq signals from WT and C /EBPβ enh −/− cells are shown at the top. f Fold change (log 2 ) in gene expression between C /EBPβ enh −/− and WT cells for genes nearest to C/EBPβ/BRD4-codepleted enhancers or super-enhancers with or without H3K27ac loss. Boxes represent 1st, 2nd, and 3rd quartiles, and whiskers show 1.5 times the interquartile range below and above the 1st and 3rd quartiles, respectively. g , h GO analyses in g super-enhancer- and h enhancer-target genes using Metascape. The length of the bars represents the level of enrichment measured as a ratio between the number of genes overlapping an MSigDB gene set over the expected frequency if such overlaps were to occur at random. i , j C/EBPβ, BRD4, and H3K27ac ChIP-seq, RNA-seq tracks and enhancer/super-enhancer calls at the i RALB and j FGFR2 loci in WT and C /EBPβ enh −/− HepG2 cells. Gene expressions determined by qRT-PCR are also shown in right. The mRNA levels were calculated by the 2 −ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average value of the WT group. Data are presented as mean ± SD. * P < 0.05; *** P < 0.001; **** P < 0.0001 as calculated by unpaired two-tailed Student’s t -test ( f – j )

Article Snippet: The primary antibodies for western blotting are CEBPB (sc-150, Santa Cruz Biotechnology, 1:1000), β-actin (8H10D10, Cell Signaling Technology, 1:10,000), Vinculin (sc-25336, Santa Cruz Biotechnology, 1:1000), H3K27ac (39133, Active Motif, 1:1000), and H3 (4499, Cell Signaling Technology, 1:1000).

Techniques: Genome Wide, Activity Assay, Western Blot, ChIP-sequencing, Binding Assay, Gene Expression, RNA Sequencing, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Liver governs adipose remodelling via extracellular vesicles in response to lipid overload

doi: 10.1038/s41467-020-14450-6

Figure Lengend Snippet: a The fold change of several miRNAs under HFD-EVs compared with Chow-EVs. b The relative miRNAs levels in miRNA knockout adipocytes that received WT, miR-122 −/− , let-7e-5p −/− , miR-31-5p −/− or miR-210-3p −/− EVs derived from primary hepatocytes. c EVs from primary hepatocytes isolated from Chow- or HFD-fed WT mice and transfected with miRNA inhibitors (Chow-EV, HFD-EV, HFD-EV-WT, HFD-EV-miR-210-3p −/− , HFD-EV-let-7e-5p −/− , HFD-EV-miR-31-5p −/− ) were added to 3T3-L1 preadipocytes. Adipocytes that differentiated from 3T3-L1 preadipocytes at day 8 after MDI induction were stained with ORO. (Scale bar, 50 μm). d Expression of genes related to adipogenesis and lipogenesis in the 3T3-L1 preadipocytes from c . e EVs from primary hepatocytes isolated from HFD-fed WT or LKO mice and transfected with various miRNA mimics (HFD-WT EV, HFD-LKO EV, HFD-LKO EV-mimic-NC, HFD-LKO EV-miR-210-3p-mimic, HFD-LKO EV-let-7e-5p-mimic, HFD-LKO EV-miR-31-5p-mimic) were added to 3T3-L1 preadipocytes. Adipocytes that differentiated from 3T3-L1 preadipocytes at day 8 after MDI induction were stained with ORO. (Scale bar, 50 μm). f Expression of genes related to adipogenesis and lipogenesis in the 3T3-L1 preadipocytes from e . g Purified PKH67-labelled EVs secreted by primary hepatocytes transfected with Cy3-labelled let-7e-5p were incubated with 3T3-L1 preadipocytes cultured in a chamber. (Scale bar, 5 μm). h Expression of genes related to fatty-acid oxidation and thermogenesis in 3T3-L1 preadipocytes cultured with or without EVs from primary hepatocytes transfected with let-7e-5p mimic. i Putative miRNA target sites of let-7e-5p within the 3′-UTR of Pgc1α. Relative luciferase activity in 3T3-L1 cells co-transfected with EV-let-7e-5p-mimic and reporter plasmid constructs containing either the WT or mutated 3′-UTR of Pgc1α. j – k Pgc1α mRNA and protein levels in 3T3-L1 cells co-transfected with plasmids expressing Pgc1α or a scrambled control (NC) and with EV-mimic-NC or EV-let-7e-5p-mimic j , or with adenovirus expressing an shRNA targeting Pgc1α (sh-Pgc1α) or a scrambled control shRNA (sh-NC) and simultaneously transfected with EV-WT or EV-let-7e-5p −/− k . Data are presented as the mean ± SEM. n = 3 biologically independent samples per group. Groups were analysed using an unpaired t test, * P < 0.05, ** P < 0.01; ## P < 0.01, sh-NC vs. sh-Pgc1α. Source data are provided as a Source Data file. See also Supplementary Figs. and .

Article Snippet: Primary antibodies targeting CD63 (Abcam, 1:1000; ab68418), TSG101 (Abcam, 1:1000; ab125011), Syntenin-1 (Abcam, 1:1000; ab205861), Rab5 (Abcam, 1:1000; ab18211), Rab27A (Abcam, 1:1000; ab55667), CD81(Santa Cruz Biotechnology, 1:1000; sc-166029), Pgc1α (Santa Cruz Biotechnology, 1:1000; sc-518038), GGPPS (Santa Cruz Biotechnology, 1:200; sc-271680), β-actin (Santa Cruz Biotechnology, 1:1000; sc-47778), Calnexin (Cell Signaling Technology, 1:1000; 2433), CALR (Cell Signaling Technology, 1:1000; 12238), H3 (Cell Signaling Technology, 1:1000; 4499) and AGO2 (Proteintech, 1:500; 10686-1-AP) were used.

Techniques: Knock-Out, Derivative Assay, Isolation, Transfection, Staining, Expressing, Purification, Incubation, Cell Culture, Luciferase, Activity Assay, Plasmid Preparation, Construct, Control, shRNA

Journal: Nature Communications

Article Title: Liver governs adipose remodelling via extracellular vesicles in response to lipid overload

doi: 10.1038/s41467-020-14450-6

Figure Lengend Snippet: Induced Ggpps expression in hepatocytes by acute and chronic HFD consumption gives rise to hepatocyte-derived EV secretion through Rab27A geranylgeranylation, which remodels adipose tissue via adipogenesis and lipogenesis. Hepatocyte-derived EVs containing miRNAs enhance lipid deposition in adipocytes by increasing lipogenesis and inhibiting lipid oxidation through the let-7e-5p-Pgc1α axis.

Article Snippet: Primary antibodies targeting CD63 (Abcam, 1:1000; ab68418), TSG101 (Abcam, 1:1000; ab125011), Syntenin-1 (Abcam, 1:1000; ab205861), Rab5 (Abcam, 1:1000; ab18211), Rab27A (Abcam, 1:1000; ab55667), CD81(Santa Cruz Biotechnology, 1:1000; sc-166029), Pgc1α (Santa Cruz Biotechnology, 1:1000; sc-518038), GGPPS (Santa Cruz Biotechnology, 1:200; sc-271680), β-actin (Santa Cruz Biotechnology, 1:1000; sc-47778), Calnexin (Cell Signaling Technology, 1:1000; 2433), CALR (Cell Signaling Technology, 1:1000; 12238), H3 (Cell Signaling Technology, 1:1000; 4499) and AGO2 (Proteintech, 1:500; 10686-1-AP) were used.

Techniques: Expressing, Derivative Assay