Fig. 7

Atr inhibition increases liver size and decreases DNA replication in uhrf1 mutant hepatocytes. (A) Representative images of γH2AX immunofluorescence and EdU labeling at 96 hpf and 120 hpf embryos following H2O2 treatment as a positive control and of uhrf1−/− mutants and sibling control livers. r-values indicate Pearson's correlation of co-localized pixels from γH2AX and EdU fluorescence. (B) Western blot of whole embryo extracts from 120 hpf uhrf1−/− and control siblings for Chk1p, RPAP, γH2AX and H3 as a loading control. (C) Quantification of the RPAP pixel intensity in 120 hpf uhrf1−/− and their respective control hepatocytes. Insets show representative images of RPAP stained hepatocytes. (D) Representative images of 120 hpf uhrf1−/− and control larvae with Tg(fabp10a:nls-mCherry) after DMSO or VE- 821 treatment, liver highlighted in red. (E) Quantification of the left liver lobe area of 120 hpf VE- 821 treated and untreated embryos from uhrf1−/− and control larvae. (F) Quantification of EdU positive cells following 30 min of incorporation at 120 hpf in DMSO and VE- 821 treated control and uhrf1−/− livers. Scale: 50 μm in A, 100 μm in C, 1000 μm in D, the number of samples and clutches indicated for each condition. P-value *< 0.05, **< 0.005, *** < 0.0005 by unpaired Student's t-test with adjustment for multiple comparisons. Data represented as median with range. (G) Model of the relationship between uhrf1 mutation, DNA methylation, cell cycle regulation, DNA replication and hepatic outgrowth failure. Uhrf1 and Dnmt1 loss causes DNA hypomethylation, upregulation of TEs and inflammatory response. This also inhibits Cdkn2a/b at protein levels, causing Cdk4/6 activation, induction of DNA replication, replication stress, DNA damage and Atr mediated dormant origin firing. In a parallel pathway, Cdk4/6 also activates Tnfa and cell death. We speculate that the DNA damage response inhibits cell division, restricting organ outgrowth and causing lethality in uhrf1 mutants.