Fig 1: Model of Kbhb under ketogenic conditionsIn ketogenic liver, as the concentration of ß-OHB rises, so does the concentration of its activated CoA form ß-OHB-CoA, which serves as the substrate for Kbhb. ß-OHB-CoA may be generated by mitochondrial enzymes that participate in fatty acid ß-oxidation or by ACSS2, a nucleo-cytoplasmic enzyme that generates other short-chain acyl-CoA species. Kbhb has the potential to feed back on the hepatic proteome and impact metabolism—as demonstrated for AHCY and the methionine cycle—as well as alter gene expression through modification of histones.
Fig 2: AHCY mediates oscillation of H3K4me3.(A) Average H3K4me3 coverage around the TSS of expressed transcripts at the two analyzed CTs. (B) Boxplots of number of H3K4me3 tags per peak within promoter regions of expressed genes (Wilcoxon signed-rank test). (C) IGV (Integrative Genomics Viewer) profile of BMAL1- and H3K4me3-enriched regions over the Dbp locus. (D) H3K4me3 ChIP at the Dbp and Per2 loci in MEFs treated with vehicle (DMSO) or DZnep (10 µM). Samples were collected at the indicated CTs, and immunoprecipitated chromatin was quantified by RT-PCR (mean ± SEM, n = 3 per time point, per group; **P = 0.01; ***P = 0.001; ANOVA, Holm-Sidak post hoc). (E) H3K9 and K14-acetyl ChIP at the Dbp and Per2 loci in MEFs treated with vehicle (DMSO) or DZnep (10 µM) (mean ± SEM, n = 3 per time point, per group; ***P = 0.001; ANOVA, Holm-Sidak post hoc). (F) H3K4me3 ChIP at the Dbp and Per2 loci in control (Ctrl) or AHCY null MEFs (KO) (mean ± SEM, n = 4 per time point, per group; **P = 0.01; unpaired Student’s t test).
Fig 3: yH2AX spread analysis. (A) Upper panel: example of three types of cell nuclei after yH2AX immunocytochemistry regarding the number of foci (red: Alexa 594 ?-H2AX foci; blue: DAPI cell nucleus). Graph: frequency distribution of nuclei with 0 to 65 foci in increments of five (calculated from 220 nuclei per cell line; p = 0.00001 determined by two tailed t-test). Imaged with Leica SP8 X FLIM confocal microscopes (HC PL APO CS2 63 × /1.40 OIL objective). (B) Primary DNA damage in HepG2 cells estimated by the alkaline comet assay. Photomicrographs of the nuclei of HepG2 cells observed after the alkaline comet assay procedure. (a) control shCTRL cells; (b) control shCTRL cells cultivated with the addition of adenosine; (c) HepG2 cells with silenced ACHY (shAHCY), where an arrow indicates the damaged DNA that resembles comet-style features; (d) HepG2 cells with silenced ACHY (shAHCY) cultivated with the addition of adenosine. Agarose microgels were stained with ethidium bromide (20 µg/mL) and analysed under epifluorescence microscope (Olympus BX51), under 200× magnification. Photomicrographs acquired by the image analysis system Comet Assay IVTM (Perceptive Instruments Ltd., UK). (C) indicators for DNA damage were (a) tail length, (b) tail intensity, and (c) total area. For each sample, three replicates were prepared, with 100 independent comet measurements per slide, with 300 measurements performed per sample. The image analysis system ‘Comet Assay IVTM’ (Perceptive Instruments Ltd., UK), in combination with an epifluorescence microscope (Olympus BX50, Japan) equipped with appropriate filters, under 200x magnification, was used for the analysis. The results are shown as the median/mean value, and the range of the measured values (min-max); scale bar, 20 µm. Statistical significance of the data was evaluated using descriptive statistics, ANOVA with post hoc Scheffé’s test (intra-group comparisons) and the Mann-Whitney U-test (inter-group comparisons). The level of statistical significance was set at P < 0.0.05. The abbreviations above the whiskers indicate which samples differ with statistical significance. For ANOVA, the abbreviations are as follows: nc – vs. corresponding negative control; # – vs. all other samples. A sign * designates the samples that showed a statistically significant increase of the studied comet parameter compared to the related clone with regard to the addition of adenosine. shAHCY.1 – cells with silenced AHCY, replica 1; shAHCY.2 – cells with silenced AHCY, replica 2., Positive control – shCTRL cells exposed ex vivo to 50 µM hydrogen peroxide for 10 minutes on ice. For each sample, three replicate slides were prepared.
Fig 4: The proposed model that connects AHCY activity, DNA damage and the regulation of the cell cycle through adenosine levels in hepatocellular carcinoma cells. Left: General schematic representation of proposed model: lowered AHCY activity causes adenosine depletion, stalling of replication forks and subsequent DNA damage, which activates various signalling pathways and causes cell cycle arrest in the G1/S checkpoint. Strong and sudden lowering of the AHCY activity causes immediate proliferation changes in cancer cells; however, mild inactivation of AHCY would cause chronic stress for liver cells and thus contribute to adult onset liver disease, such as hepatocellular carcinoma as observed in the latest case of AHCY deficiency. Right: A detailed overview of how adenosine depletion could cause replication fork stalling through misbalance of the dNTP pool due to lower dATP levels, and subsequent impairment of adequate rates of DNA synthesis and progression of the replication forks. Treatment with hydroxyurea, although following another pathway, facilitates a similar and well-described effect through the disturbance of the balance of the total dNTP pool.
Fig 5: AHCY is important for amplitude of circadian oscillation.(A) Circadian bioluminescence traces for Bmal1:luc U2OS cells transfected with either siRNA control (siCtrl) or siRNA targeting Ahcy (siAhcy) (mean values shown, n = 4). (B) Bar graph of relative circadian amplitude of U2OS Bmal1:luc siCtrl and siAhcy cells (mean ± SEM, n = 4; *P = 0.05; unpaired Student’s t test). (C) Heatmaps representing genes cyclic in control MEF cells only (Ctrl; 261 genes) and in both conditions (16 genes) (n = 3 per time point, per group; P < 0.01 in each dataset). Pie chart representing the percentage of genes oscillating in both conditions and in control only. (D) Gene Ontology (GO) term enrichment analysis of genes oscillating in the control group only. (E) Transcription factor binding site (TFBS) analysis of rhythmic transcription factors (TFs) on transcripts rhythmic exclusively in control MEFs. Represented as percentage of TFBS. (F) Bar graph of amplitude analysis of clock genes in control (Ctrl) and AHCY null (KO) MEF cells. (G) Heatmap of hierarchical clustering of genes significantly down-regulated or up-regulated in vehicle-treated MEFs (DMSO) between the two analyzed CT points (CT12 and CT24; n = 3 per time point per group; fold change > 2; FDR < 0.05). Heatmap shows gene expression levels of control (DMSO) and DZnep-treated MEFs. (H) GO term enrichment analysis of genes down-regulated or up-regulated in vehicle-treated MEFs (DMSO) between the two analyzed CT points (CT12 and CT24). (I) Pie chart showing the percentage of time-dependent genes identified in (G) (715 genes) differentially expressed in MEFs treated with DZnep (10 µM). (J) Bar graph displaying log2 fold change of gene expression of genes related to (G). (K) Bar graph of fold change analysis of clock genes in vehicle (DMSO)– and DZnep-treated MEF cells. ***P = 0.001; unpaired Student’s t test.
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