Fig. 6

Response capability of cardiac IREs requires sufficient AP-1 or ETS motif frequency. A Bar plots showing Hif1α mRNA expression level under normal and hypoxic conditions as measured by qPCR, using Gapdh for normalization. Error bars denote SEM (N = 3). P-value: unpaired Student’s t-test (**, P < 0.01). B Western blot for HIF1α protein levels collected from HL-1 cells under normal and hypoxic conditions, using GAPDH for negative control. C Genome browser view showing H3K27ac ChIP-seq signals at the genomic regions around two mouse-specific IREs (E1 and E2) in mouse uninjured (red) and regenerating (blue) samples, as well as corresponding orthologous sequences in zebrafish. D Bar plots showing mRNA relative expression level of Pde1a (left) and Gda (right) under normal and hypoxic conditions, using Gapdh for normalization. Error bars denote SEM (N = 3). P-value: unpaired Student’s t-test (*, P < 0.05). E Schematic of IRE sequences without mutation, and with ETS or AP-1 motif mutations (orange: ETS motif, yellow: AP-1 motif, grey: mutated sequence). Values of E1 and E2 length are listed below the graph. F Luciferase reporter assay for wildtype, ETS or AP-1 motif mutated mouse IREs under normal (orange) and hypoxic (blue) conditions. Empty vectors are used for negative control. Error bars denote SEM (N = 4). P-values: unpaired Student’s t-test (*, P < 0.05; **, P < 0.01; n.s., not significant). G Lollipop plot showing activation levels quantified by fold change of average relative luciferase activity from (D) of two mouse IREs without mutation and with ETS or AP-1 motif mutations upon hypoxia, using the empty vector for normalization. H A model summarizing the inter-species variations between zebrafish and mouse IREs and their associated genes. Left, AP-1 and ETS motif frequencies of shared IREs in zebrafish and mice are similar, and nearby genes are up-regulated upon injury. Middle, orthologous sequences of zebrafish-specific IREs in mice are not activated by injury with reduction in motif frequency, and nearby genes show no changes in expression level. Right, for mouse-specific IREs, orthologous sequences in zebrafish are with decreased ETS motif frequency, but nearby genes are still with upregulation, which is mediated by IRE-independent mechanisms. Red pentagrams stand for ETS motifs, and the blue for AP-1 motifs. Rectangles with solid line indicate IREs identified through H3K27ac ChIP-seq data analysis, and rectangles with dotted line represent orthologous sequences of IREs. Black rectangles are IRE-associated genes or orthologs in the other species