R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation

Abstract

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11–q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are imprinted neurodevelopmental disorders caused by oppositely inherited deficiencies of chromosome 15q11–q13. AS and PWS are both characterized by hypotonia at birth, disordered sleep, autistic features, and intellectual disabilities, but the diseases differentiate into phenotypically distinct syndromes in early childhood (1, 2). Seizures, ataxia, and inappropriate laughter characterize AS, whereas hyperphagia leading to obesity and obsessive-compulsive behaviors characterize PWS. Maternal mutations in UBE3A/Ube3a in humans and mice have identified the loss of function of this ubiquitin E3 ligase encoding gene as the cause of AS (3, 4). For PWS, small deletions of the HBII-85/SNORD116 locus (5–7) and two mouse models of Snord116 deletions (8, 9) have identified the minimal causative deficiency to be the paternally expressed, highly repetitive, long noncoding RNA (lncRNA) that is processed into multiple small nucleolar RNAs (snoRNAs) and spliced nuclear retained host genes (116HG and 115HG) (10, 11).

A recent drug screen discovered that topoisomerase inhibitors, including topotecan, reduce Ube3a-ATS by an unknown mechanism to reverse the silencing of paternal Ube3a in mouse neurons and brain (12). Topotecan activates paternal Ube3a by reducing the abundance of an antisense transcript, Ube3a-ATS, at the 3′ end of a 1-Mb paternal transcript originating at the PWS imprinting control region (PWS-ICR; Fig. 1A) (13). Ube3a-ATS is an atypical RNA polymerase II transcript that is not polyadenylated and silences paternal Ube3a in a transcription-dependent manner, most likely by transcription through the locus (14). Such a mechanism of imprinted transcriptional silencing from an antisense lncRNA has also been observed at the imprinted Airn antisense to the growth factor receptor encoding Igf2r (15). Ube3a-ATS is expressed only in neurons, although transcription initiating at the PWS-ICR occurs in all tissues to give rise to ubiquitously expressed Snrpn. Regulation of Ube3a-ATS appears to occur through alternative transcription termination potentially related to transcription initiating at upstream U-exons, although the mechanism has not been identified (16, 17). In nonneuronal cells, transcription terminates within the Snord116 region, but in neurons, transcription continues through the Snord116 region to generate Ube3a-ATS and silence paternal Ube3a (14). Reversing paternal UBE3A silencing with topotecan holds promise as a therapy for AS because it would compensate for loss of maternal UBE3A; however, concerns about specificity persist (18).

Fig. 1.

Deletion of the Snord116 locus mitigates topotecan mediated inhibition of Ube3a-ATS. (A) Schematic of Snrpn to Ube3a with genes, primer locations, RNA FISH probe locations, and location of deletion in Snord116del mouse. Paternal and maternal alleles indicated by Pat and Mat, respectively. (B) qRT-PCR of RNA isolated from 10 d in vitro cultured neurons normalized to Gapdh and graphed relative to WT mock treated levels. (C) Percentage of nuclei positive for Ube3a-ATS RNA FISH on 10 d in vitro cultured neurons from WT and Snord116del mice mock treated or treated with 300 nM topotecan. % reduction labeled, and graphed in Fig. S1. (D) Results for RNA FISH using the Ube3a sense probe graphed as % nuclei exhibiting biallelic (two spots), monoallelic (one spot), or nonexpressing (no spots) pattern of Ube3a Representative images of Ube3a-ATS (E and F) or Ube3a (G and H) RNA FISH in WT neurons (Left) or Snord116del neurons (Right) treated with or without 300 nM topotecan. White arrows indicate RNA FISH signals (red). Nuclei stained with DAPI (blue). Results are graphed as mean ± SEM from four biological replicates of 150–200 nuclei each. *P < 0.05, **P < 0.01, ***P < 0.001. n.d., not detectable.

Topotecan acts as an inhibitor of topoisomerase I (19), but the mechanism by which it specifically represses Ube3a-ATS is unknown. Topoisomerase activity is necessary to relieve the topological strain caused by transcription (20). Loss of topoisomerase activity in front of the elongating RNA polymerase leads to increased supercoiling and inhibition of transcription (21), whereas loss of topoisomerase activity behind the RNA polymerase complex leads to melting of the DNA and the formation of DNA:RNA hybrids called R loops (22). R loops form at CpG island promoters, the 3′ ends of genes, and recombination sites (23–25). The formation of R loops slows RNA polymerase by increasing the forces impeding transcriptional progression including steric hindrance caused by wrapping of the RNA (26, 27).

Here we demonstrate that R-loop formation occurs over repetitive intronic segments of high GC skew in mouse Snord116 and human SNORD116 PWS loci and that this genetic locus mediates the effects of topotecan treatment on Ube3a-ATS expression. Topetecan increased formation of R loops and chromatin decondensation over the active paternal Snord116 allele and stalled transcriptional progression through Ube3a-ATS. These results improve understanding of the relationship between imprinting, chromatin dynamics, and transcriptional regulation of the imprinted PWS/AS locus that will inform more targeted therapies for these and other related autism-spectrum disorders.

Results

To determine the genetic requirement for the Snord116 PWS locus in mediating topotecan action, neural precursor cell (NPC) cultures from mice with paternal deletion of Snord116 (Snord116del) and wild-type (WT) littermates were differentiated to neurons and treated with 300 nM topotecan using conditions that effectively activate paternal Ube3a in mouse neurons (12). Steady-state transcript levels assayed by quantitative reverse transcriptase PCR (qRT-PCR) demonstrated significantly increased Ube3a-ATS transcript levels in Snord116del compared with WT neurons, as well as expected deficiencies in host gene transcripts 116HG and IPW with no significant change in Snrpn and Ube3a transcript levels (9) (Fig. 1B). RNA fluorescence in situ hybridization (FISH) using a probe designed to detect Ube3a-ATS showed that 80–90% of Snord116del neurons expressed Ube3a-ATS, compared with only 25% of WT neurons (Fig. 1C, E, and F). Topotecan treatment of neurons derived from WT NPCs resulted in an 86% reduction in the number of cells showing Ube3a-ATS signal and an 89% reduction in Ube3a-ATS transcript levels, as expected (12) (Fig. 1 B, C, and E and Fig. S1). In contrast, the Snord116 deletion diminished the inhibitory effect of topotecan treatment on Ube3a-ATS expression, with only 39% reduction in the number of Ube3a-ATS positive cells, and minimal decrease in Ube3a-ATS transcript levels (P = 0.0546) (Fig. 1 B, C, and F). No significant change in Snrpn transcript levels was observed with topotecan treatment, suggesting that topotecan acts between Snrpn and Ube3a-ATS within the Snord116 region (Fig. 1B). Although Ube3a transcript levels measured by qRT-PCR were unchanged with topotecan treatment, RNA FISH using a Ube3a sense-specific probe demonstrated an expected increase in the number of nuclei showing two Ube3a signals (Fig. 1 D and G), confirming the derepression of the paternal allele shown previously (12). In contrast, there was no associated change in the number of cells expressing Ube3a biallelically with Snord116 deletion (Fig. 1 D and H). Two-color RNA FISH using probes to detect 115HG and sense Ube3a RNA revealed that the increase in biallelic expression in WT neurons with topotecan treatment coincided with a loss of 115HG expression, suggesting that RNA polymerase is not progressing beyond the 116HG locus in cells that reexpress paternal Ube3a (11) (Fig. S1). ChIP-qPCR analysis provided supportive evidence that topotecan treatment reduced the progression of RNA Pol-II downstream of Snord116 (Fig. S2). Together, these results demonstrate that the paternal Snord116 locus is required for optimal topotecan-driven inhibition of Ube3a-ATS. Furthermore, because transcript levels were unaffected at the Snrpn 5′ end, these results indicate that topotecan acts primarily within the Snord116 locus.

We previously demonstrated that the paternal Snrpn-Ube3a chromatin becomes decondensed in a transcription-dependent and DNase-sensitive manner in postnatal neurons (28). Because topotecan reduces transcription of Ube3a-ATS, we investigated its effect on the chromatin structure of the maternal and paternal alleles by performing DNA FISH for the Snrpn-Ube3a locus on WT and Snord116del neurons. As previously shown, the paternal allele was decondensed relative to the maternal allele (Fig. 2A). Topotecan specifically and significantly increased the decondensation of the paternal allele in WT neurons (Fig. 2 A and C). In contrast, topotecan did not change the length of the decondensed paternal allele in Snord116del neurons (Fig. 2 A and D). DNA FISH analysis of the Snord116 locus demonstrated that the increased decondensation with topotecan treatment occurs primarily at Snord116 (Fig. 2 B and E). Chromatin decondensation measured by DNA FISH was neuron-specific (Fig. 2 E and F), corresponding to tissue-specific differences in nucleosome occupancy at Snord116 in vivo measured by chromatin immunoprecipitation (ChIP) for total histone H3 (Fig. S2). These results show that Snord116 is required for topotecan-mediated chromatin decondensation at the Snrpn-Ube3a region, and thus that Snord116 is the specific topotecan-responsive region.

Fig. 2.

Topotecan treatment increases chromatin decondensation of the Snord116 region. (A) DNA FISH signal length of the entire Snrpn-Ube3a region on paternal (decondensed) and maternal (condensed) alleles in 10 d in vitro cultured neurons from WT and Snord116del mice that were treated with or without 300 nM topotecan. (B) DNA FISH measurements of shorter Snrpn-Snord116 region in WT mice, showing topotecan effect on chromatin decondensation at Snord116. (C and D) Representative images of DNA FISH in WT (C) or Snord116del (D) neurons treated without (Upper) or with (Lower) topotecan. (E) Combined DNA FISH of Snrpn-Ube3a (red) and Snrpn-Snord116 (green) in an adult mouse neuron with nucleus outlined showing overlap on paternal decondensed allele of longer and shorter FISH probes. (F) Adult mouse liver nucleus using the same combined FISH probes as E but the lack of chromatin decondensation. Paternal decondensed allele (P) is shown in each neuronal image. (Scale bar: 1 μm.) Nuclei counterstained by DAPI (blue). Results are graphed as mean ± SEM from two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

To identify potential sequence characteristics of the Snord116 region that could explain the specific nature of topotecan activity, we quantified the distribution of G and C bases across the two DNA strands, a property called GC skew (Fig. 3A). In both mouse and human, the introns in the Snord116 repeat region showed positive GC skew on the transcribed strand (Fig. S3). This GC skew pattern is conserved from mouse to human, even though the sequence itself does not show conservation as measured using phyloP or phastCons (Fig. S3). Positive GC skew on the transcribed strand of a GC-rich region is predictive of the potential to form cotranscriptional R loops (Fig. 3B) (23). R loops, which are structurally more similar to A-form DNA than to the typical B-form DNA:DNA duplex (29), have been shown to prevent nucleosome binding in vitro (30). In addition, R loops have negative effects on transcription elongation (22, 24, 27), and inhibition of topoisomerase activity has been shown in yeast and human cells to lead to increased R-loop formation (22, 31). Due to the G-richness of the transcribed strand in the Snord116 repeat region and the stabilizing effect of topoisomerase inhibition on R loops, we hypothesized that increased R-loop formation was responsible for the effects of topotecan on this locus.

Fig. 3.

The mouse Snord116 region gives rise to a G-rich primary RNA and R-loop formation depletes nucleosomes. (A) GC skew (red, +50% to −50%) is mapped over the Snrpn to Ube3a locus together with exon locations and GC-percent in the UCSC genome browser. (B) Diagram showing cotranscriptional R-loop formation with a G-rich sequence (red) but not a C-rich sequence (blue). (C) A control R-loop forming region shows decreased plasmid supercoiling upon in vitro transcription that disappears with RNase H treatment (Upper) and evidence for R loops by dot blot (Lower). DRIP confirms R-loop formation when G-rich RNA is produced, but not C-rich RNA. (D and E) A cloned G-rich intron from mouse Snord116 (D) and human SNORD116 (E) showed a change in plasmid supercoiling (Upper) and R-loop detection by dot blot (Lower) when in vitro transcribed, which were diminished by RNase H treatment. DRIP confirms that Snord116 and SNORD116 form R loops upon transcription induction in cells. (F) A transcribed region shows a decrease in nucleosome occupancy when a G-rich RNA is produced, but not a C-rich RNA. (G) Cloned mouse Snord116 shows a decrease in nucleosome occupancy upon induction of transcription. Results are graphed as mean ± SEM from three replicates (DRIP) or six replicates (ChIP). *P < 0.05, **P < 0.01, ***P < 0.001.

To test for R-loop formation at a GC-skewed locus and at Snord116, we cloned a control R-loop forming region (mouse Airn; ref. 23) in the physiologic (G-rich RNA) or nonphysiologic (C-rich RNA) orientation and intronic Snord116 repeat regions from mouse and human genomic DNA into in vitro transcription vectors and inducible episomal vectors (Fig. S4). R-loop formation upon in vitro transcription can be detected as an RNase H-sensitive gel-shift of the plasmid from the supercoiled state (Fig. 3C, arrowhead) to the relaxed state (Fig. 3C, arrow). The presence of R loops can be confirmed by immunoblotting analysis with the S9.6 DNA:RNA-specific antibody (23) (Fig. 3C). Similar to the control Airn region, cloned genomic regions from mouse Snord116 and human SNORD116 exhibited R-loop formation with in vitro transcription by gel-shift analysis and immunoblotting (Fig. 3 D and E). Episomes are stably replicating, chromatinized extrachromosomal DNA entities that can be used to measure R-loop formation by DNA:RNA immunoprecipitation (DRIP) followed by qPCR (DRIP-qPCR) using the S9.6 antibody. Here, R loops were enriched only when transcription was induced through the control Airn region in the physiologic (G-rich RNA) orientation (Fig. 3C). In contrast, the nonphysiologic (C-rich RNA) orientation did not result in R-loop formation, nor did a constitutively expressed control region (Fig. 3C). Episomes carrying regions cloned from mouse Snord116 and human SNORD116 also showed R-loop formation by DRIP upon induction of transcription in the physiologic orientation (Fig. 3 D and E). The presence of a G-skewed sequence was inhibitory to transcription because, under similar culture and induction conditions, a C-skewed sequence induced more readily, compared with a G-skewed sequence and exhibited leaky read-through transcription in the absence of Doxycycline induction (Fig. S4).

Having established that R loops form when transcription was induced on episomes, we performed H3 ChIP to measure the effect that R-loop formation may have on nucleosome occupancy. Coinciding with orientation-specific R-loop formation, H3 ChIP showed a significant decrease in nucleosome occupancy with the induction of transcription only when the transcribed region was in the physiologic orientation (Fig. 3F). Confirming that R-loop formation on the Snord116 intronic sequence leads to loss of nucleosome occupancy, H3 ChIP of the Snord116 episome showed depletion of nucleosomes relative to a negative control region when transcription was induced (Fig. 3G). Together, these results indicate that R-loop formation at Snord116 can cause transcriptionally dependent chromatin decondensation of this locus.

For further evidence of R-loop formation at Snord116 in vivo, we performed DRIP-qPCR on genomic DNA isolated from WT adult mouse cerebellum. DRIP-qPCR revealed R-loop formation at the Snord116 locus and GC skewed positive controls, but not at an unskewed locus (Fig. 4A). In addition, combined immunofluorescence with the S9.6 R-loop antibody and DNA FISH to Snrpn-Ube3a showed that the two signals colocalized at the decondensed paternal allele (Fig. 4B and Fig. S5). In addition, topotecan treatment of neurons resulted in increased R-loop formation detected by DRIP at all loci, but R-loop formation was greater at Snord116 compared with three control loci (Fig. 4C). The combined results of these analyses demonstrate that R loops form under physiologic conditions at Snord116, but topotecan stabilizes increased R-loop formation at Snord116.

Fig. 4.

The endogenous Snord116 region forms R loops in neurons and topotecan increases R-loop formation at Snord116. (A) DRIP of cerebellum DNA reveals R-loop formation at Snord116, and two positive control loci (Nfe2l1 and Actn4), but not a negative control region (AK132462). (B) Combined immuno-FISH reveals R-loop formation on the decondensed paternal allele. (Scale bar: 1 μm.) (C) DRIP of DNA from topotecan and mock treated neurons shows increased R-loop signal at Snord116, but not three other loci. (D) A model for topotecan inhibition of topoisomerase I actions leading to stabilization of R-loop formation in Snord116 leading to chromatin decondensation and mediating topotecan inhibition of Ube3a-ATS. Results are graphed as mean ± SEM from four biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

Much more is known about the mechanism of transcriptional initiation regulation at the promoters of genes compared with regulation of transcription progression (32), particularly for long transcripts such as Snrpn-Ube3a. In this study, we provide evidence for R-loop formation in G-rich repeats of the Snord116 locus causing displacement of nucleosomes in a transcription-dependent manner corresponding to chromatin decondensation of the paternal allele. Our previous study had shown that transcription on the paternal allele was necessary for chromatin decondensation to occur, but did not identify a specific mechanism (28). The Snord116 locus likely has a propensity for forming R loops because of the repeating units of GC skew, thus explaining the particular sensitivity of the region to inhibition of topoisomerase I by topotecan (12). We demonstrate that topotecan treatment results in increased R-loop formation within Snord116 corresponding to increased chromatin decondensation and inhibition of transcriptional elongation through to Ube3a-ATS (Fig. 4D). Although the topotecan effects on R-loop levels observed by DRIP appear to show a smaller effect than those seen at the transcriptional levels, we hypothesize that the repetitive nature of the Snord116 locus amplifies the effect of R loops individually forming at each repeat. These results predict that R-loop formation at the Snord116 locus finely balances the chromatin state and transcription at the PWS/AS locus. Under physiologic conditions, R-loop formation and chromatin decondensation enable the transcription machinery to progress through to Ube3a-ATS to silence Ube3a in cis. However, under conditions that promote excess R-loop formation, such as topotecan treatment, R-loop formation and the resulting topological strain cause excessive stalling of the transcription machinery thereby preventing expression of Ube3a-ATS (Fig. 4D). The repetitive nature of the Snord116 locus may contribute to the sensitivity of the region to topotecan because GC skew is present across the ∼100-kb Snord116 repeat region (Fig. 3A). R-loop formation may also play a role in the physiologic regulation of Ube3a-ATS expression because R loops have been shown to form at the 3′ end of genes and contribute to transcription termination (24). Maintaining the optimal amount of R-loop formation and transcription in the locus will be important for therapies in AS or PWS that seek to alter transcription on the paternal or maternal allele to reverse the disease state.

Regulation of transcription termination in the Snord116 region is part of normal neuron development because the Snord116 region and Ube3a-ATS are expressed specifically in neurons, whereas Snrpn is expressed ubiquitously from the same promoter. Our finding that the Snord116 region forms R loops in neurons under physiologic conditions, but that excessive R-loop formation terminates transcription, suggests that regulation of R-loop formation may be one mechanism by which transcription regulation occurs in the Snrpn-Ube3a locus. R loops have been shown to be part of transcription termination at the 3′ end of genes (24). Furthermore, inhibiting RNA processing events can lead to increased R-loop formation (33, 34). Although such a role underlies genomic instability, we hypothesize that regulation of alternative splicing and processing in the Snord116 region could also modulate R-loop–mediated transcription termination in the locus.

UBE3A-ATS serves as an appealing target for therapy in AS because paternal UBE3A is intact in AS patients (35). The discovery of topoisomerase inhibitors as potential therapies for silencing Ube3a-ATS and activating paternal UBE3A is very promising (12), but concerns about toxicity and specificity remain (18). Our findings on the mechanism and site of action of topotecan in repressing paternal Ube3a-ATS may lead to refined strategies for reducing unwanted off-target effects of topoisomerase inhibitors in treating AS. We show that the Snord116 locus is a critical region for mediating the effects of topoisomerase inhibition on Ube3a-ATS expression. Because inhibiting RNA processing can increase R-loop formation (33, 34), one strategy may be to target splicing and RNA processing of the Snord116 and 116HG transcript to inhibit transcription leading to Ube3a-ATS.

Materials and Methods

NPC and Neuron Culture.

B6(Cg)-Snord116^tm1.1Uta/J (Snord116del) mice were obtained from Jackson Labs, housed in a 24-h light/dark cycle, temperature-controlled room, and fed a standard diet of Picolab mouse chow 20 (PMI International). NPCs were isolated from E15.5 mouse brain (36) and grown in NEP Complete media without serum supplemented glutamax. For differentiation, media was changed to NEP Basal with retinoic acid, and 50% of media was replaced every 3.5 d. For topotecan treatment of neurons, after 7 d in vitro, 300 nM topotecan (Sigma) was added, and neurons were grown for 3 more days.

Episome and Plasmid Construct.

Genomic DNA was PCR amplified and ligated into the pGEM-T vector (Promega). Sequence-confirmed inserts were subsequently cloned between CMV-TetOn promoter and IRES-GFP sequences. mAirn G-skew and C-skew sequences were characterized (23). The mouse Snord116 episome carries a single 1,050-bp fragment most closely matching chr7:67,007,613–67,008,662 (mm9, 97% identity). The human Snord116 episome carries a single 1,087-bp fragment matching chr15:25,299,483–25,300,569 (hg19).

Episome Culture.

HEK-293 cells stably maintaining episomes were grown in selective media (200 μg/mL Hygromycin) and treated with 1 μg/mL doxycycline to induce transcription. RNA or chromatin was harvested 48 h later.

RNA Extraction.

RNA was extracted from neurons using manufacturer’s protocol for RNeasy mini kit (Qiagen).

DNA FISH.

DNA FISH was performed as described (28).

RNA FISH.

Cells grown on coverslips were fixed in Histochoice for 15 min, washed in 1× PBS, and stored in 70% (vol/vol) EtOH at −20 °C. Slides were dehydrated in 100% EtOH and probe resuspended in SureFISH hybridization buffer (Agilent) was applied. Hybridization was performed overnight at 37 °C before washing coverslips twice in 2× SSC/0.1% IGEPAL at 37 °C and mounting in SureFISH mounting buffer (Agilent) with DAPI at 5 μg/mL using novel custom probes for Ube3a, as well as 115HG and 116HG probes, described previously (11).

Microscopy.

Microscopy was performed as described (28). Two individuals blind to the conditions analyzed DNA FISH and RNA FISH slide. For RNA FISH, four biological replicates (150–200 nuclei per replicate) were analyzed. For DNA FISH, two biological replicates (40 nuclei per replicate) were analyzed. Two-tailed Student’s T test was performed to test for significance.

qRT-PCR and qPCR.

qRT-PCR and qPCR were performed as described (37) with custom primers listed in Table S1. Two-tailed Mann–Whitney u test was used to test for statistical significance of qRT-PCR. RNA was analyzed from six biological replicates.

For GC skew maps, GC skew was generated using a custom Perl script to calculate within a 500-bp window the G-skew defined as ((G − C)/(G + C)).

Immunostaining.

Immunostaining for RNA:DNA hybrids was performed as described (28) using the S9.6 antibody (23). For RNase H treatment of slides, after postfixation, slides were incubated in 1× RNase H buffer with 10 units of RNase H (NEB).

Immuno-FISH.

For immuno-FISH slides were prepared as for immunostaining above. After staining, slides were washed in 1xPBS/0.2% tween, fixed in 1% formaldehyde, and washed three times in 1× PBS. Slides were dehydrated in a series of 50%/70%/90%/100% EtOH washes, and hybridization was performed as for DNA FISH.

DRIP.

For DRIP, genomic DNA (gDNA) was extracted from cultured neurons as described (23). gDNA was extracted from one hemisphere of a mouse cerebellum by homogenizing in 1× PBS, and nuclei were pelleted and resuspended in R-loop lysis buffer. Episomal DNA was extracted by resuspending cells in 10 mM EDTA/10 mM Tris⋅Cl (pH 7.5) before lysing cells with 0.5% SDS and pelleting gDNA with 1.25 M NaCl at 4 °C overnight. gDNA and Episomal DNA was digested with HinDIII, EcoRI, SspI, BsrGI, and XbaI. DRIP was performed as described (23) with 8 μg of DNA and 10 μg of antibody. DRIP on topotecan treated neurons was performed on four biological replicates and two-tailed Mann–Whitney u test was used for statistical testing. Cerebellum DRIP was performed on three biological replicate samples, and two-tailed Student t test was used for significance testing.

ChIP.

Chromatin immunoprecipitation was performed as described (37) using an antibody for total histone H3 (Abcam ab1791) or RNA PolII 8WG16 (Abcam ab817). Brain and liver nucleosome occupancy was compared from three animals and two-tailed Student t test was used for significance testing. Pol-II ChIP was performed on three biological replicates. Episome DRIP and ChIP was performed on six biological replicates and two tailed Mann–Whitney u test was used for significance testing.

IVT R-Loop Gel Shift.

Plasmids were in vitro transcribed with T7, Sp6, or T3 RNA polymerase (Promega) for 30 min and heat inactivated at 65 °C for 5 min. Sample was treated with RNase H (NEB) or mock treated. DNA was precipitated with ethanol and washed before agarose gel electrophoresis stained with Sybr Gold (Invitrogen).

Dot Blot R-Loop Assay.

Plasmids were in vitro transcribed as for the gel-shift assay. Purified plasma DNA (250 ng) was blotted on a membrane and cross-linked to the membrane with UV. The membrane was blocked with Odyssey blocking buffer (Odyssey) for 30 min, and incubated with 5 μg of S9.6 antibody. Blots were washed in 1× PBS/0.2% Tween-20, and DAMIG-800 in blocking buffer (Odyssey) was added at room temperature for 2 h. The membrane was washed with 1× PBS/0.2% Tween and scanned on a Licor Odyssey scanner.