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. 2022 Feb 22;11:e69476. doi: 10.7554/eLife.69476

Figure 1. 5-Hydroxymethylcytosine (5hmC) favors co-transcriptional R-loop formation.

(A) Native or modified deoxyribonucleotides (dCTPs) were incorporated upon PCR amplification into DNA fragments with sequences from the transcription termination region of ACTB (ACTB P1 and ACTB P2) or APOE. (B) Incorporation of dCTP variants confirmed by immunoblotting using specific antibodies against 5-methylcytosine (5mC), 5hmC, and double-stranded DNA (dsDNA). (C) R-loops formed upon in vitro transcription reactions were detected by immunoblotting using the S9.6 antibody. RNase H-treated in vitro transcription reaction products (RH+) serve as negative controls. All data are representative of seven independent experiments with similar results. (D) S9.6 immunoblots were quantified and the R-loop levels normalized against the levels detected in the reaction products of DNA templates containing native C. Data represent the mean and standard deviation (SD) from seven independent experiments. *p<0.05, **p<0.01, and ***p<0.001, two-tailed Student’s t-test. (E) In vitro transcription reaction products of ACTB P2 templates were visualized using atomic force microscopy (AFM). R-loop structures obtained from 5hmC-containing ACTB P2 transcription in the absence (RH-) or presence (RH+) of RNase H are shown. R-loops present in the transcription reaction products of C, 5mC, or 5hmC-containing ACTB P2 templates were counted in a minimum of 80 filaments observed in three individual AFM experiments.

Figure 1.

Figure 1—figure supplement 1. Cytosine modifications do not affect the detection of DNA:RNA hybrids by the S9.6 Ab.

Figure 1—figure supplement 1.

(A) DNA:RNA hybrids formed through annealing of C, 5-hydroxymethylcytosine (5hmC), or 5-methylcytosine (5mC)-containing ssDNA with the complementary ssRNA. Data are representative of three independent experiments. (B) Electrophoretic mobility shift assay (EMSA) of DNA:RNA hybrids incubated with increasing amounts of the S9.6 Ab in a native polyacrylamide gel. Data are representative of three independent experiments. (C) Quantification of free DNA:RNA hybrids. The graph shows means and SD from three independent experiments.
Figure 1—figure supplement 2. In vitro transcription levels of different DNA templates.

Figure 1—figure supplement 2.

(A) RNA concentration obtained from in vitro transcription of DNA templates containing C, 5-hydroxymethylcytosine (5hmC), or 5-methylcytosine (5mC). Data shown are the mean from three independent experiments. (B) Mean and SD of the RNA levels obtained in each reaction normalized to reactions using DNA templates with native cytosines. Data are from three independent experiments.