GADD45A binds R-loops and recruits TET1 to CpG island promoters

Abstract

R-loops are DNA-RNA hybrids enriched at CpG islands (CGIs) that can regulate chromatin states^1-8. How R-loops are recognized and interpreted by specific epigenetic readers is unknown. Here we show that GADD45A (growth arrest and DNA damage protein 45A) binds directly to R-loops and mediates local DNA demethylation by recruiting TET1 (ten-eleven translocation 1). Studying the tumor suppressor TCF21 (ref. ⁹), we find that antisense long noncoding (lncRNA) TARID (TCF21 antisense RNA inducing promoter demethylation) forms an R-loop at the TCF21 promoter. Binding of GADD45A to the R-loop triggers local DNA demethylation and TCF21 expression. TARID transcription, R-loop formation, DNA demethylation, and TCF21 expression proceed sequentially during the cell cycle. Oxidized DNA demethylation intermediates are enriched at genomic R-loops and their levels increase upon RNase H1 depletion. Genomic profiling in embryonic stem cells identifies thousands of R-loop-dependent TET1 binding sites at CGIs. We propose that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs.

Figure 1. TARID forms an R-loop at the TCF21 promoter.

a. DRIP-qPCR analysis at the TCF21 promoter in the indicated cell lines transfected with plasmids encoding RNase H1 (RNH1 +) or control GFP (-). Scheme illustrates the TCF21 locus and the position of amplicons used in qPCR. b. RT-qPCR in cells transfected with plasmids encoding GFP (Ctrl), RNH1, or the hybrid-binding (HB) domain of RNH1. RNA levels were normalized to HPRT1 mRNA. c. R-loop footprinting showing PCR products of bisulfite-converted DNAs from PSF and H387 cells. Diagram depicts amplicons (a-d), position of unmodified primers (black), and primers matching bisulfite-modified DNA (red) used for PCR. d. Native bisulfite sequencing of single-stranded DNA showing TARID-R-loop position at the TCF21 transcription starting site (see also Supplementary Fig. 1d). Cartoon indicating in scale the position of cytosines (all C) and of Cs converted to T residues (R-loop position) in the region analyzed. e. Ectopic TARID forms an R-loop at TCF21. DRIP-qPCR in H387 cells transfected with TARID full-length (red), exon2 (green), or intronic control RNA (blue). RPL13A, R-loop positive control. f. TARID induces demethylation of the TCF21 promoter. MassARRAY DNA methylation analysis in H387 cells transfected with TARID variants. The position of CpG residues is indicated by open lollipops. Methylation level is scaled from 0 to 1 and plotted for each CpG residue in the amplicon. Panels a,b,e,f: mean ± s.d., n = 3 biological replicates, two tailed t-test; *P<0.05, **P<0.01, *** P<0.001. Panels c,d: Experiments were repeated once with similar results. Blot image was cropped (see Supplementary Fig. 9a).

Figure 2. GADD45A binds R-loop structures in vitro and in vivo.

a. ChIP-qPCR of GADD45A at the TCF21 promoter in PSFs overexpressing RNase H1 (RNH1) or GFP. Data are normalized to control IgGs. Mean ± s.d., n = 3 biological replicates, two tailed t-test; *P<0.05. b. Pull-down assay showing GADD45A binding to R-loops in vitro. Bead-bound S9.6 antibody or GADD45A (GA45a), PTB or GFP were incubated with radiolabeled R-loops followed by RNase A or RNH1 treatment. Bound probes were visualized by PhosphorImaging. c. EMSA showing binding of GADD45A to R-loops. A radiolabeled R-loop probe (upper panel) or RNA oligonucleotide (lower panel) was incubated with increasing amounts of GADD45A, followed by PAGE and PhosphorImaging. d. Competitive EMSA showing concentration-dependent displacement of GADD45A from R-loops by DNA:RNA hybrids (upper panel) but not by dsDNA (lower panel). e. Quantitation of competitive EMSAs using a GADD45A/R-loop complex and the indicated unlabeled competitors. GADD45A binding to R-loops was set to 1. f. Northwestern blot showing binding of membrane-bound GADD45A to R-loops in vitro. Where indicated, the membrane was treated with RNase A or RNase A plus RNase H1. The asterisk marks a nonspecific band in the mock sample. g. GADD45A and TET1 are associated with cellular R-loops. Lysates from PSFs expressing FLAG-GADD45A (GA45a upper panel) or HA-TET1 and HA-CBP (lower panel) were immunoprecipitated with S9.6 antibody and co-precipitated proteins were visualized on Western blots. Where indicated, lysates were treated with RNase H1 (RNH1) before immunoblotting. Panels b-g: Experiments were repeated twice with similar results. All blot images were cropped (see Supplementary Figs. 9b-d and 10a-b).

Figure 3. Transcription of TARID precedes R-loop formation and TCF21 activation.

a. RT-PCR analysis of TARID and TCF21 mRNA in FACS-sorted PSFs. RNA levels were normalized to HPRT1 mRNA. b. DNA methylation at the TSS of TCF21 measured by MassARRAY in FACS-sorted cells. Methylation is plotted as mean value of all informative CpG residues in the amplicon scaled from 0 to 1. c. TARID (red) and TCF21 mRNA (blue) levels in PSFs arrested at the G₁/S boundary and released into the cell cycle by thymidine removal. RNA levels are normalized to HPRT1 mRNA. d. DRIP-qPCR in synchronized PSFs as in c. e. ChIP-qPCR monitoring GADD45A occupancy at the TCF21 promoter in synchronized PSFs. The levels are normalized to control IgGs. f. RT-qPCR monitoring TET1 mRNA in synchronized PSFs. RNA levels were normalized to HPRT1 mRNA. g. Analysis of 5hmC-modified TCF21 promoter in synchronized PSFs. 5hmC levels at CpG #7 of the TCF21 promoter (see Supplementary Fig 3b) measured by quantitative hydroxymethylcytosine analysis. h. ChIP-qPCR monitoring HA-TET1 occupancy (left) and relative levels of 5hmC at the TCF21 promoter (right) in FACS-sorted PSFs expressing HA-TET1. i. Analysis of 5hmC at the TCF21 promoter in synchronized PSFs harvested 12h following transfection with RNaseH1 (RNH1) or GFP. j. Schematic of peaks of TARID expression, R-loop formation, and TCF21 expression proceeding sequentially during cell cycle progression. Panels a-i: mean ± s.d., n = 3 biological replicates; h-i two tailed t-test; *P<0.05, **P<0.01, *** P<0.001.

Figure 4. R-loops recruit TET1 to CGI promoters.

a. LC-MS/MS analysis of genomic levels of modified cytosines after siRNA-mediated knockdown of RNH1 in control or HEK293T cells expressing HA-TET1. b. LC-MS/MS analysis of genomic levels of modified cytosines in inputs or S9.6 enriched R-loop fractions from abdomen (Abn) and head (Hd) of three mouse embryos at stage E15.5. c. Differential enrichment TET1 ChIP-seq analysis of RNH1- vs. mock-treated mESCs samples (n=3, biological replicates). The MA plot depicts 90,482 consensus TET1 peaks, RNH1-decreased peaks (red), and RNH1-increased peaks (blue) (FDR<0.01). d. Metagene profiles of average TET1 ChIP-seq read coverage over all protein coding genes e. Peak overlap of all vs. RNH1-sensitive TET1 peaks with CpG islands (CGI), Transcription Start Sites (TSS), and published R-loops. f. UCSC browser screenshot of one RNH1-sensitive TET1 peak associated with Gadd45a TSS and a TARID-like lncRNA (E230016M11Rik). Other tracks show CGI, R-loops, and CpG methylation. A broader view of this locus with all ChIP-seq samples is shown in Supplementary Fig. 6e. g. ChIP-qPCR monitoring GADD45A occupancy at RNase H1-sensitive TET1 peaks in the absence (Mock) or presence of RNase H1 (RNH1) along with negative controls. Data are normalized to control IgGs. h. ChIP-qPCR monitoring TET1 binding in human primary skin fibroblasts at promoters of human orthologs of the genes shown in (g) upon treatment with GADD45A- and GADD45B-specific siRNA or scrambled control siRNAs. Panels a,b,g,h : mean ± s.d., n = 3 (g,h n=4;) biological replicates, two tailed t-test; *P<0.05, **P<0.01, *** P<0.001.