cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool
- University of Miami Miller School of Medicine, United States
- University of California, San Francisco, United States
- University of California, Berkeley, United States
- Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, United States
- Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, United States
Figures
Figure 1 with 4 supplements
cAMP induces 5hmC in cells.
(A) Dot-blot shows that treatment with ascorbate (50 μM) for 3 days or cAMP (100 μM) for 7 days induced 5hmC in Schwann cells. (B) Semi-quantification of the dot-blot shows that both cAMP and …
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Figure 1—source data 1
Primers used for quantitative RT-PCR.
- https://doi.org/10.7554/eLife.29750.007
Figure 1—figure supplement 1
cAMP induces 5hmC in different cell types.
cAMP (10 μM) induced 5hmC in HEK-293 cells, MEF, and SH-SY5Y cells after treatment for 8 hr. Scale bar = 20 μm.
Figure 1—figure supplement 2
5hmC elevation by shorter treatment of forskolin in Schwann cells.
(A) Schwann cells were treated with forskolin (10 μM) for 3 hr followed by washout. 5hmC induction was detected at of 0, 3, and 24 hr time points following treatment. Cells continuously treated with …
Figure 1—figure supplement 3
5hmC elevation by shorter treatment of cAMP and forskolin in HEK-293 cells.
(A) HEK-293 cells were treated with cAMP (10 μM) for 1 or 4 hr followed by washout. 5hmC was detected at the time point of 24 hr, which is at a level comparable to the continuous treatment (24 hr). …
Figure 1—figure supplement 4
Treatment with cAMP does not increase Tet transcripts.
qRT-PCR shows that Tet1 mRNA remained at a similar level (p=0.478) after treatment of Schwann cells with cAMP (100 μM) for 1 day. In contrast, levels of Tet2 mRNA (p=0.001) and Tet3 mRNA (p=0.0001) …
Figure 2 with 3 supplements
cAMP increases the intracellular labile Fe(II) pool in cells.
(A) cAMP (1–100 μM) treatment for 4 hr increased the intracellular labile Fe(II) pool detected by Trx-Puro ferrous iron probes. (B) IF quantification shows the dose-dependent effect of cAMP on …
Figure 2—figure supplement 1
Negative control for puromycin incorporation with labile Fe(II) probe TRX-puro in Schwann cells.
(A) No immunofluorescence (IF) signal was observed after incubation with negative control dioxolane compound, Diox-Puromycin, (1 μM) for 2 hr, indicating no puromycin incorporation. Strong IF signal …
Figure 2—figure supplement 2
cAMP increases the intracellular labile Fe(II) pool in different cell types.
cAMP (10 μM) also enhanced the intracellular labile Fe(II) pool detected by Trx-Puro probes in HEK-293, MEF, and SH-SY5Y cells after treatment for 4 hr. Scale bar = 20 μm.
Figure 2—figure supplement 3
(A) Representative ratiometric confocal microscopy images of live HEK-293 cells loaded with FIP-1.
Scale bar = 20 μm. (B) Quantification of mean Green/FRET ratios, which represent the relative abundance of labile Fe(II) in the cell. Statistical significance was assessed by calculating p-values …
Figure 3 with 3 supplements
Induction of labile Fe(II) by endogenous cAMP and the dependency of 5hmC generation on labile Fe(II).
(A) AMP (100 μM) treatment for 4 hr did not induce labile Fe(II) while AC activators (forskolin (100 μM), bicarbonate (50 mM)) and PDE inhibitors (caffeine (100 μM), IBMX (100 μM)) increased labile …
Figure 3—figure supplement 1
Relatively transient increase intracellular labile Fe(II) by short forskolin treatments.
Schwann cells were treated with forskolin (10 μM) for 3 hr followed by washout and Fe(II) detection by Trx-Puro probe. Intracellular labile Fe(II) elevation was observed at time points of 0, 3, and …
Figure 3—figure supplement 2
Ascorbate does not increase the intracellular labile Fe(II) pool in Schwann cells detected by Trx-Puro probes.
Treatment of Schwann cells with 50 μM sodium ascorbate for 4 hr caused no obvious change in labile Fe(II). In comparison, immunofluorescence shows an increase of labile Fe(II) in cells after …
Figure 3—figure supplement 3
Pretreatment with iron chelators 2,2, bipyridyl (20 μM) and deferoxamine (20 μM) for 20 min blocked the upregulation of 5hmC by cAMP (100 μM) treatment in A2058 cells.
Scale bar = 20 μm (n = 2 independent experiments with three biological replicates each).
Figure 4 with 1 supplement
cAMP increases labile Fe(II) and 5hmC by enhancing the acidification of endosomes.
(A) cAMP (100 μM) treatment decreases the pH in intracellular vesicles. Red fluorescence increases when the pH decreases from 8 to 4. (B) V-ATPase inhibitor Bafilomycin A1 (200 nM) pretreatment for …
Figure 4—figure supplement 1
Decreasing the expression of Ferritin does not block the induction of labile Fe(II) by cAMP.
(A) cAMP (100 μM) treatment induces labile Fe(II) detected by Trx-Puro probes at a comparable level in Ferritin heavy chain knockdown cells compared to the cells treated with scramble siRNA or with …
Figure 5 with 3 supplements
The regulation of labile Fe(II) by cAMP is likely mediated via RapGEF2.
(A) Pretreatment with PKA inhibitors (KT5720 (2 μM), H89 (20 μM)), Epac inhibitor ESI09 (10 μM) or CNGC blocker LCD (10 μM) for 20 min prior to cAMP addition showed no effect on labile Fe(II) …
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Figure 5—source data 1
Fragments per kilobase per million (FPKM) of CNG and Rapgef genes in Schwann cells.
- https://doi.org/10.7554/eLife.29750.022
Figure 5—figure supplement 1
Abolishment of PKA activity by inhibitors H89 and KT5720.
(A) Pretreatment with H-89 (20 μM) and KT5720 (2 μM) decreased the band of phosphorylated Peptag peptide induced by cAMP (100 μM) treatment. (B) Quantification of the assay showing that H89 (20 μM) …
Figure 5—figure supplement 2
qRT-PCR shows that the mRNA level of RapGEF2 is lower in the siRNA group compared to the scramble siRNA group (p=0.042) (n = 3 independent experiments with three biological replicates each, error bars denote standard error).
https://doi.org/10.7554/eLife.29750.020
Figure 5—figure supplement 3
RAP1 and labile Fe(II) induction by cAMP.
(A) Knocking down the expression of Rap1 largely blocked the effect of cAMP (100 μM) on vesicle acidification and labile Fe(II) elevation detected by Trx-Puro probes, while knocking down RAP2 and …
Figure 6
GPCR stimulation induces labile Fe(II) and 5hmC generation in Schwann cells.
(A) Treatment with Gs-coupled receptor ligands isoproterenol (10 μM) and CGRP (100 nM) for 2 days induced 5hmC, an effect comparable to treatment with cAMP (100 μM) as shown by IF. (B) IF …
Figure 7
cAMP shifts the transcriptome and hydroxymethylome of Schwann cells.
(A) cAMP (100 μM) treatment for 7 days changes genome-wide transcription as shown by the heatmap of the relative abundance of reads for differential transcripts. (B) Venn Diagram of the RNA-seq …
Figure 8 with 1 supplement
5hmC profile changes correlate with gene transcription.
(A) cAMP (100 μM) upregulated 5hmC at promoter and gene body regions of differential transcripts. (B) 33.8% of differential transcripts were associated with both 5hmC peaks and PKA-dependent …
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Figure 8—source data 1
Impact of cAMP treatment on both transcription and 5hmC levels of myelin-related genes in Schwann cells.
- https://doi.org/10.7554/eLife.29750.027
Figure 8—figure supplement 1
Analysis of hMeDIP-seq enrichment levels in the promoter region (3,000 bp upstream of TSS) and gene body regions (TSS to TES) of genes that change in response to cAMP treatment and nondifferential genes.
Overall average levels of 5hmC enrichment in read counts per million mapped reads (RCPM). Blue shading indicates promoter regions while green shading indicates gene body region.
Author response image 1
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29750.028
