We then evaluated whether etoposide-mediated early-response gene expression is a result of the activation of the DNA damage response. Cultured primary neurons pre-incubated with a specific inhibitor (KU55933; ATMi) against ATM (ataxia telangiectasia mutated) caused a marked reduction in DSB signaling, as indicated by a reduction in the intensity of γH2AX, a marker of DSB signaling ( Figure 1 E). However, ATM inhibition had no effect on the etoposide-mediated increase in Fos and Npas4 expression ( Figure 1 E). In addition to this, we tested whether treatment of neurons with other DSB-inducing agents also induced the expression of early-response genes. These agents included the radiomimetic drugs, neocarzinostatin (ncs) and bleomycin (bleo), and the PARP inhibitor, Olaparib (PARPi). Interestingly, none of these drugs recapitulated the effects of etoposide on Fos and Npas4 expression ( Figure 1 F). Together, our results unexpectedly revealed that Topo II-mediated DSBs stimulate the expression of early-response genes.

Based on these observations, we directly assessed the expression of various neuronal activity-regulated genes after etoposide treatment of cultured primary neurons using quantitative real-time PCR (qRT-PCR). Early-response genes, including Fos, FosB, Npas4, and Egr1 were all upregulated within 20 min of treatment with etoposide ( Figure 1 D). However, other activity-regulated genes, such as Bdnf and Homer1, showed no such increase in either the RNA-seq or the qRT-PCR experiments ( Figures 1 C and 1D). Thus, etoposide selectively induces a small subset of activity-regulated genes.

With the idea of further characterizing the consequences of DSB formation in neurons, we incubated cultured primary neurons with etoposide for 6 hr and performed gene expression profiling using next-generation RNA-sequencing (RNA-seq). Etoposide is an established inhibitor of topoisomerase II (Topo II) that traps the enzyme in a complex with the cleaved DNA and thereby converts a normal physiological reaction into a potentially toxic DSB. Transcriptomic analysis after etoposide treatment revealed 692 genes that were differentially expressed compared to vehicle-treated controls. Consistent with the expectation that DSBs would interfere with transcription, an overwhelming majority (680 genes) of the differentially expressed genes were downregulated ( Figure 1 A and Table S1 ). Remarkably, however, the 12 genes that were upregulated were enriched for neuronal activity-regulated genes and particularly the so-called early-response genes, such as Fos, FosB, and Npas4—transcription factors that are also rapidly expressed in response to neuronal activity ( Figures 1 B and 1C).

(F) Cultured primary neurons were incubated with the indicated drugs for 20 min, following which the expression of Fos and Npas4 was assessed using qRT-PCR (n = 3, ∗∗ p < 0.01, one-way ANOVA).

(E) Cultured primary neurons were pre-incubated with the ATM inhibitor, KU55933 (ATMi), for 30 min following which etoposide treatment was performed as in (D). (Top) Neurons were immunostained with antibodies against γH2AX following treatment with etoposide either in the presence or absence of ATMi. (Bottom) The expression of early-response genes, Fos and Npas4, were assessed using qRT-PCR (n = 3, ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA).

(D) Cultured primary neurons were treated with either etoposide (5 μM) or vehicle (DMSO) for 20 min, following which RNA was extracted, and the expression of the indicated genes were assessed using qRT-PCR (n = 3, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed t test).

(C) UCSC genome browser snapshots of RNA-seq trace files from etoposide-treated neurons (green) and vehicle-treated controls (black) at various neuronal activity-regulated genes (violet bars). y axis represents signal intensity and the scale is indicated in parentheses.

(A) Cultured primary neurons (DIV 10) were incubated with either vehicle (DMSO) or etoposide (5 μM) for 6 hr, following which RNA was extracted and subjected to RNA-seq. (Top) Differentially expressed genes are shown in a volcano plot, and genes whose expression was altered significantly (p < 0.05) are indicated in red. (Bottom) Schematic indicating the number of downregulated (blue) and upregulated genes (red).

Our results stemmed from some unexpected observations made while studying the effects of DSB formation in neurons. The accrual of DNA damage has been linked to various neurological disorders, and we previously described the formation of DNA lesions, particularly DNA DSBs, to be an apical neurotoxic event in several mouse models of neurodegeneration ().

In addition to this, mouse acute hippocampal slices that were either bath-incubated in NMDA solution or subject to theta-burst electrical stimulation also showed increased γH2AX levels compared to untreated controls ( Figures S1 D and S1E). To understand whether DSBs are also formed following neuronal activity in vivo, we subjected wild-type C57BL/6 mice to a training paradigm for contextual fear conditioning, following which we prepared hippocampal lysates and measured γH2AX levels. Similar to our observations with cultured primary neurons and hippocampal slices, elevated γH2AX levels were detectable in hippocampal lysates within 15 min after exposure to the fear-conditioning paradigm ( Figure S1 F). Moreover, DSB formation has also been reported in other neuronal stimulation paradigms (). Thus, neuronal activity correlates with the formation of DNA DSBs in neurons.

Because early-response genes are normally induced in response to stimulation of neuronal activity, we tested whether established paradigms of neuronal stimulation are also associated with DSB production. Brief incubations of cultured primary neurons with potassium chloride (KCl), N-methyl-D-aspartate (NMDA), or bicucullin (bic), all caused a substantial increase in Fos and Npas4 mRNA ( Figures S1 A and S1B). Interestingly, each of these treatments also caused an increase in the levels of γH2AX, an established marker of DNA DSBs ( Figure S1 C) ().

(F) (Top) Two month-old C57BL/6 mice were placed in a contextual chamber for 2 min, following which two 0.8 mA footshocks were delivered from the grid floor. The animals remained in the chamber for an additional 30 s and were then moved back to their home cages. Fifteen minutes following the footshock, animals were sacrificed and the hippocampus was dissected and flash frozen at −80°C. (Bottom) Lysates were then prepared and γH2AX levels were assessed by western blotting. (Right) Quantification of γH2AX levels relative to GAPDH (n = 6 animals per group, ∗ p < 0.05, two-tailed t test).

(E) Acute hippocampal slices were prepared as in (D) and subjected to 3xTBS (theta-burst stimulation), following which extracts were prepared and assessed for γH2AX levels by western blotting.

(D) (Top) Acute hippocampal slices were prepared from two month-old male C57BL/6 mice. The slices were bath incubated with NMDA (50 μM) for 5 min, followed by recovery in NMDA-free conditions for an additional 10 min. (Bottom) Extracts were then prepared from the slices and γH2AX levels were assessed using western blotting. (Right) Quantification of γH2AX levels relative to GAPDH (n = 3, ∗ p < 0.05, two-tailed t test).

(C) Cultured primary neurons were treated with KCl, NMDA, or Bic as in (A) and (B) and electrophoresed through 15% SDS-PAGE gels. Levels of γH2AX and H3 were assessed using western blotting.

(B) Cultured primary neurons were incubated with either 10 mM KCl or 50 μM NMDA. KCl treatment was for 20 min, whereas NMDA treatment was for 10 min, followed by recovery for an additional 10 min in NMDA-free media. RNA was then extracted and Fos and Npas4 expression was assessed as in (A) (n = 3, ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA).

(A) Cultured primary neurons were incubated with either 55 mM KCl or 50 μM bicucullin (Bic) for 30 min. RNA was then extracted and Fos and Npas4 expression was assessed using qRT-PCR (n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA).

A closer examination of γH2AX distribution at these loci, including at the early-response genes, revealed a peculiar pattern, with γH2AX peaks initiating adjacent to the transcriptional start site (TSS), spreading into the gene body, and terminating downstream of the 3′UTR ( Figure 2 D and Figure S2 B). In fact, γH2AX peak width was strictly proportional to gene length ( Figure S2 C). The only exception to this scenario was the case of Homer1, in which γH2AX signals only spanned across the annotated short isoform of Homer1 ( Figure S2 B). While the significance of this is presently unclear, it is noteworthy that only the short isoform of Homer1 is regulated by neuronal activity (). In contrast to these early-response genes, none of the other late response genes, including Bdnf, Rgs2, Nrn1, and Gpr3 showed an increase in γH2AX intensity in their vicinity following NMDA treatment ( Figure 2 D and Figure S2 B). Together these results indicate that activity-dependent stimulation of neurons results in the formation of DNA DSBs at very specific locations in the genome, particularly near early-response genes.

To further characterize the distribution of enriched γH2AX signals within genomic regions, we performed differential peak calling ( Experimental Procedures ) and observed that γH2AX enrichment following NMDA treatment occurs primarily within gene bodies compared to distal intergenic regions ( Figure 2 B). Surprisingly, our analysis revealed only 21 regions that were enriched for γH2AX signals in the NMDA-treated samples compared to controls. Twenty of these regions were within genes, whereas one site was detected in intergenic regions ( Figure 2 C). Remarkably, included within these 21 loci were the early-response genes, Fos, FosB, Npas4, Egr1, Nr4a1, and Nr4a3 ( Figure 2 C). In addition to the transcription factors categorized as early-response genes, several other transcription factors, Olig2 and Dlx6os1, as well as several non-coding RNAs, including Malat1, AI854517, and C130071C03Rik were also represented among the loci that showed elevated γH2AX ( Figure 2 C). Additionally, the loci identified by differential peak calling also displayed the highest γH2AX intensities in the NMDA-treated samples, but not in vehicle-treated samples ( Tables S2 and S3 ).

For an initial assessment of genome-wide γH2AX ChIP-seq signals, we classified genomic regions into 14 distinct chromatin states associated with regulatory regions (promoters, enhancers, heterochromatin, etc.) based on available ChIP-seq data of chromatin marks, including H3K36me3, H4K20me1, H3K4me1, H3K27ac, H3K4me3, H3K27me3, and H3K9me3 ( Figure S2 A) (). Correlating the positional information of these chromatin marks with raw γH2AX ChIP-seq signals revealed that the observed increase in γH2AX is confined largely to actively transcribed genes and their downstream regions, but not to enhancers, polycomb repressed regions, or heterochromatin ( Figure 2 A).

(D) UCSC genome browser views depicting the disposition of γH2AX signals within various activity-regulated genes under basal conditions (control) and following NMDA treatment. y axis represents intensity and the range is indicated in parentheses.

(C) Differential peak calling was performed to determine the regions that were enriched for γH2AX following NMDA treatment ( Experimental Procedures ). This data were then processed using CIRCOS software () to generate the shown circular representation. The outer ring depicts the mouse chromosomes. The blue ring represents a map of gene densities, and the green ring indicates γH2AX signals. Red lines within the green ring represent loci that were enriched for γH2AX relative to controls. Twenty loci were within genes, and these genes are indicated. One locus was within intergenic regions.

(B) γH2AX ChIP-seq signals enriched in NMDA-treated samples relative to controls were processed using CEAS (Cis-Regulatory Element Annotation System) program ( http://liulab.dfci.harvard.edu/CEAS/ ). (Left) Pie chart depicting the relative proportions of the indicated annotated regions in the genome. (Right) Disposition of γH2AX signals within these annotated genomic regions.

(A) Genomic regions were categorized into 14 distinct chromatin states based on combinatorial patterns of various chromatin marks ( Figure S2 A). The percentage of γH2AX peaks within each chromatin state were then normalized to the proportion of the genome with that chromatin state and plotted.

(C) Peak widths of γH2AX regions were plotted as a function of gene length of the loci that incur these signals following treatment of cultured primary neurons with NMDA. Pearson coefficient and p values for the distribution are indicated within the graph.

(B) UCSC genome browser views depicting the disposition of γH2AX signals within various genes (violet bars) under basal conditions (control) and following NMDA treatment. y axis represents signal intensity with range indicated in parentheses.

(A) Genomic regions were categorized into 14 distinct chromatin states based on combinatorial patterns of the indicated chromatin marks (TssA – active transcription start site; TssU – upstream of transcription start site; TssD – downstream of transcription start site; Tx- transcribed region; Tx3p – 3′ transcribed region; Gene – gene region, not necessarily transcribed; EnhG – Enhancer within gene region; Enh1 – Very active enhancer; Enh2 – Moderate enhancer; Bival – bivalent region (active and polycomb repressed regions); Repr. PC – polycomb repression; Het – heterochromatin; LowG – weak gene signal; LowI – intergenic) ().

To further understand the relationship between neuronal activity and DSB formation, we next determined the positions of activity-dependent DSBs on a genome-wide level. DSB formation results in the rapid phosphorylation of the histone variant, H2AX, at Ser139 in the vicinity of DSB sites (). The identification of chromatin enriched for γH2AX can be exploited to derive the locations of DSBs (). We therefore stimulated cultured primary neurons by briefly incubating them with 50 μM NMDA and then performed genome-wide γH2AX ChIP-seq.

Based on Topo IIβ binding in the promoters of early-response genes, we then speculated that if Topo IIβ generates DSBs in these promoters in an activity-dependent manner, that such cleavage should prevent the amplification of these regions using the primers that detect Topo IIβ enrichment ( Figure 3 E). Control Topo IIβ ChIP experiments following etoposide treatment of cultured primary neurons revealed a significant reduction in amplification by two distinct primer sets that span the Fos promoter ( Figure 3 F). Importantly, Topo IIβ ChIP following NMDA treatment also indicated a sharp reduction in the amplification of the Fos promoter regions ( Figures 3 F and S3 D). A similar attenuation in PCR amplification was also observed following ELK1 ChIP in NMDA-treated neurons ( Figure 3 G). To determine whether activity-induced DNA cleavage occurs at a specific site within the Fos promoter, we repeated the PCR amplification assays after Topo IIβ ChIP using various primer combinations that spanned the originally identified Topo IIβ binding region within the Fos promoter. However, each region that showed enriched amplification following Topo IIβ ChIP under basal conditions also showed reduced amplification following NMDA treatment ( Figure S3 E). Thus, activity-induced DSBs are not site-specific but rather occur broadly within the Fos promoter. Taken together, our results indicate that neuronal activity-induced DSBs occur within the promoters of early-response genes and that these DSBs could result from the activities of Topo IIβ.

We next performed DNA cleavage assays in which we incubated purified, recombinant Topo IIβ together with supercoiled plasmids that either contained or lacked the upstream sequences of the Fos gene. As expected, incubation of supercoiled plasmids with Topo IIβ caused an increase in relaxed (Form II) DNA ( Figure 3 D). In the presence of etoposide, an increase in linear (Form III) DNA was also detected in plasmids containing Fos upstream sequences. However, the generation of this linear DNA was sharply attenuated when Fos upstream sequences were deleted ( Figure 3 D). Similar results were also observed with plasmids containing Npas4 upstream sequences. Together, these results suggest that Topo IIβ preferentially cleaves the promoters of early-response genes.

To test whether Topo IIβ activity could underlie activity-induced DSB formation, we first measured levels of Topo IIβ cleavage complexes following NMDA treatment using an immune complex of enzyme (ICE) assay (). As a control, we incubated neurons with etoposide and observed an increase in Topo IIβ covalent complexes ( Figure S3 C). Interestingly, NMDA treatment also caused an increase in Topo IIβ covalent complexes, indicating that neuronal activity results in Topo IIβ-mediated DNA cleavage ( Figure S3 C). To further assess how neuronal activity affects Topo IIβ, we immunoprecipitated Topo IIβ following NMDA treatment of primary neurons and incubated the precipitated Topo IIβ with supercoiled plasmids. Incubation with Topo IIβ caused a potent relaxation of supercoiled plasmids in both NMDA-treated and vehicle-treated samples ( Figure 3 C). However, plasmids incubated with Topo IIβ from NMDA-treated samples also displayed increased smearing, indicative of fragmented DNA ( Figure 3 C). These results suggest neuronal activity confers Topo IIβ with an increased propensity to generate DNA breaks.

The regulatory elements that bind the Fos promoter are well characterized, and additional ChIP experiments revealed that Topo IIβ co-occupies the Fos promoter together with ELK1, as well as the lysine deacetylase, HDAC2 ( Figure 3 B and Figure S3 A). We previously showed that HDAC2 binds to the promoters of several early-response genes and negatively modulates their expression (). While screening for proteins that could potentially regulate HDAC2 binding to these promoters, we separately discovered that the protein, tyrosyl DNA phosphodiesterase 2 (TDP2), binds HDAC2 and is enriched at several early-response gene promoters, including Fos and Npas4, under basal conditions ( Figure S3 B and data not shown). These results are intriguing because Topo II-mediated DSBs involve the formation of a covalent phosphotyrosyl bond between Topo II and the DNA, and TDP2 specializes in processing these intermediates and in the repair of Topo II-mediated DSBs ().

(E) ChIP analysis of Topo IIβ binding to various regions within the Fos promoter under basal conditions (control) and following NMDA treatment. (Top) Relative positions of various primers upstream of the TSS are indicated in parentheses. (Bottom) ChIP analysis using the indicated primer sets. The βglobin promoter was also probed as a control (n = 4, ∗∗ p < 0.01, two-way ANOVA).

(D) ChIP analysis of Topo IIβ binding to the indicated regions following either etoposide or NMDA treatment as in Figure 3 F.

(C) Cultured primary neurons were treated with either ETP or with NMDA as before, following which the cells were lysed using 1% sarkosyl. Lysates were then layered onto CsCl gradients (4 ml) and centrifuged for 12 hr at 71,000 rpm in a TLA-100.3 rotor. Pellets were washed, resuspended in TE buffer, and the indicated amounts of DNA were applied to nitrocellulose membrane using a slot-blot apparatus (Bio-Rad). The adsorbed DNA was then probed for Topo IIβ using standard western blotting protocols.

(A) ChIP analysis of ELK1 binding to the promoters of Fos and Npas4. Two distinct primer sets (Fos Prom#1 and Fos Prom#2) were used to probe the Fos promoter region. In addition, two different exons within the Fos gene (exons 2 and 4), as well as the Npsa4 and β-globin promoters were also probed (n = 3, ∗ p < 0.05, one-way ANOVA).

We began by assessing Topo IIβ binding at various regions of the prototypical early-response gene, Fos, under basal conditions. Topo IIβ ChIP-qPCR indicated negligible binding in the exons of the Fos gene, as well as within the promoters of β-globin and GAPDH ( Figure 3 A). However, Topo IIβ binding was significantly enriched within the Fos promoter ( Figure 3 A). Similarly, Topo IIβ binding was also enriched within the Npas4 promoter ( Figure 3 A). These results suggest that Topo IIβ is bound to the promoters of early-response genes under basal conditions.

(F) ChIP analysis of Topo IIβ binding at the Fos promoter following either etoposide or NMDA treatment. Control bar graphs are as in (A) (n = 3, ∗ p < 0.05, ∗∗∗ p < 0.001, two-way ANOVA).

(E) Schematic showing how DNA cleavage by Topo IIβ (red ovals) would preclude the amplification of the Fos promoter by PCR primers utilized in (A) (indicated by blue and green arrows).

(D) Luciferase reporter constructs containing sequences upstream of either the Fos TSS or the Npas4 TSS were incubated with purified recombinant human Topo IIβ (8 units/reaction) either in the presence or absence of etoposide (0.2 mM final). Reactions were then incubated at 30°C for 15 min, stopped, and electrophoresed through 1% agarose gels. As controls, constructs lacking the Fos and Npas4 sequences (ΔFos-luc and ΔNpas4-luc) were also analyzed. Dashed line indicates the size of the linearized construct. Letters indicate the positions of supercoiled (I), relaxed (II) and linear (III) DNA.

(C) Topo IIβ was immunoprecipitated from cultured primary neurons following NMDA treatment. The precipitated Topo IIβ was then incubated with 1 μg of a supercoiled luciferase reporter plasmid carrying ∼6 kb of upstream regions of the Npas4 gene. Reactions were then incubated at 30°C for 15 min, stopped, and electrophoresed through 1% agarose gels. Letters indicate the positions of supercoiled (I) and relaxed (II) DNA. Substrate DNA alone was run to indicate the migration of supercoiled and relaxed DNA (top). Input fractions (5%) collected prior to immunoprecipitation were electrophoresed through 6% SDS-PAGE gels and analyzed by western blotting (bottom).

(B) Sequential ChIP analysis of HDAC2 and Topo IIβ binding to the Fos promoter. Cultured primary neurons were first subjected to ChIP with antibodies against Topo IIβ. The crosslinked proteins were then immunoprecipitated with antibodies against HDAC2. Primers were as in (A) (n = 3, ∗ p < 0.05, one-way ANOVA).

(A) ChIP analysis of Topo IIβ binding to the promoters of Fos and Npas4. Two distinct primer sets (Fos Prom#1 and Fos Prom#2, respectively) were used to probe the Fos promoter region. In addition, two different exons within the Fos gene (exons 2 and 4), as well as the promoters of Npas4, β-globin, and GAPDH, were also probed (n = 3, ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA).

In an effort to determine the mechanisms that underlie the formation of neuronal activity-induced DSBs, we returned to our original observation that etoposide treatment is specifically able to upregulate the expression of early-response genes ( Figure 1 F). Etoposide introduces DSBs by targeting Topo II (). Mammalian cells express two distinct isoforms of Topo II, Topo IIα, and Topo IIβ. Topo IIα is mainly expressed in dividing cells, whereas Topo IIβ is robustly expressed in postmitotic cells, including neurons, and is primarily implicated in transcription-related functions (). Incidentally, Topo IIβ-mediated DSBs were previously shown to be essential for estradiol-stimulated activation of gene expression (). These observations caused us to focus specifically on Topo IIβ.

To further understand whether Topo IIβ-mediated DNA cleavage could underlie the selective pattern of DSB formation in response to neuronal activity, we performed γH2AX ChIP-seq after treatment of cultured primary neurons with etoposide. Similar to results with NMDA treatment, γH2AX signals after etoposide treatment were enriched primarily within gene bodies compared to intergenic regions ( Figure 4 E). Furthermore, aggregate plots revealed that etoposide-induced and NMDA-induced γH2AX signals display a strikingly similar distribution pattern at the sites of activity-induced DSBs ( Figures 4 F and S4 A), and this similarity is further emphasized from the comparison of NMDA and etoposide-induced γH2AX signals at individual genes ( Figure S4 B). These data suggest that Topo IIβ-mediated DNA cleavage could underlie activity-induced DSB formation in neurons.

(B) UCSC genome browser views depicting the disposition of γH2AX signals within various genes (violet bars) following either etoposide or NMDA treatment compared to controls. The γH2AX signal intensities after NMDA are as in Figures 2 D and S2 B. y axis represents signal intensity with range indicated in parentheses.

(A) Peak widths of γH2AX regions were plotted as a function of gene length of the loci that incur these signals following treatment of cultured primary neurons with etoposide. Pearson coefficient and p values for the distribution are indicated within the graph.

Interestingly, NMDA caused a substantial increase in genome-wide Topo IIβ binding. Whereas 430 Topo IIβ peaks were identified under basal conditions, 2,416 peaks were detected within 20 min of the initial NMDA treatment ( Table S4 ). This nearly 5-fold increase in Topo IIβ signals was largely proportionally distributed within the same chromatin regions that showed Topo IIβ binding under basal conditions ( Figure 4 A and Table S4 ). NMDA treatment also caused an enrichment of Topo IIβ signals at SRF, CREB, and CBP binding sites ( Figures 4 B and 4C). These features also extend to the promoters of early-response genes, such as Fos and Npas4, where an increase in Topo IIβ signals were clearly detected following neuronal activity ( Figure 4 D). Importantly, Topo IIβ signals under both basal and NMDA-treated conditions were found immediately adjoining γH2AX-enriched regions in NMDA-treated samples ( Figure 4 D). These results further suggest that Topo IIβ binding patterns are consistent with their role in activity-dependent DNA DSB formation.

To further examine the connections between Topo IIβ and activity-induced DSBs, we performed Topo IIβ ChIP-seq under basal conditions and following neuronal activity. An analysis of Topo IIβ signals within various genomic regions suggested that under basal conditions, Topo IIβ binding is enriched primarily at and upstream of the TSS of actively transcribed genes, as well as at enhancer elements, but not at heterochromatin or polycomb repressed regions ( Figure 4 A and Table S4 ). A comparison with existing datasets of various factors that bind activity-regulated genes () revealed that Topo IIβ binding is enriched at SRF and CREB binding sites, as well as CBP binding sites within promoters and enhancers ( Figures 4 B and 4C). These results further indicate that Topo IIβ binding patterns overlap with regulators of neuronal activity-induced gene expression.

(F) Aggregate plots of input-normalized γH2AX signals at the 20 loci that show increased γH2AX intensity following NMDA treatment were generated for NMDA-treated (orange), etoposide-treated (blue) and control (gray) conditions. Graph on the left shows the distribution within a 2 kb window of the transcription start site (TSS), whereas the graph on the right denotes the distribution near the transcription termination site (TTS). Plots were generated using annotatePeaks.pl command of HOMER software and custom R scripts.

(E) γH2AX ChIP-seq signals enriched in etoposide-treated samples relative to controls were processed using CEAS (Cis-Regulatory Element Annotation System) program ( http://liulab.dfci.harvard.edu/CEAS/ ). (Left) Pie chart depicting the relative proportions of the indicated annotated regions in the genome as in Figure 2 A. (Right) Disposition of differential γH2AX signals within these annotated genomic regions following etoposide treatment.

(D) UCSC genome browser views denoting the disposition of γH2AX and Topo IIβ signals at Fos and Npas4 under the indicated conditions. y axis denotes signal intensity and the range is indicated in parentheses.

(C) Binding profiles of Topo IIβ within a 4 kb window of CBP peaks at either promoters (left) or enhancers (right) were generated as in (B).

(B) Publicly available ChIP-seq datasets of SRF and CREB () were used to determine the binding profiles of Topo IIβ relative to the binding sites of these proteins. The graphs indicate the averaged binding patterns of Topo IIβ within a 4 kb window of all SRF (left) and CREB (right) peaks. Dashed line indicates the profile under basal conditions, whereas the solid line depicts the profiles following NMDA treatment.

(A) Genomic regions were categorized into 14 distinct chromatin states based on combinatorial patterns of various chromatin marks ( Figure S2 A). The percentage of Topo IIβ peaks within each chromatin state were then normalized to the proportion of the genome within that chromatin state and plotted.

To determine whether the association between Topo IIβ and CTCF is relevant to activity-induced DSBs, we again utilized the publicly available CTCF ChIP-seq datasets and examined the disposition of CTCF relative to γH2AX signals at the sites of activity-dependent DNA DSBs. Our analysis revealed that the probability of finding a CTCF peak within 2 kb of the boundary of γH2AX regions was significantly greater than the probability of finding permutated random sites in the mappable regions throughout the genome (p < 0.0001). Furthermore, CTCF peaks lie significantly closer to the TSS of genes that incur DSBs following NMDA treatment compared to other genes ( Figure S5 E). Aggregate plots of CTCF and γH2AX distribution at sites of activity-induced DSBs further revealed that CTCF peaks tightly envelop γH2AX regions within these loci ( Figure 5 E). Similar results were also observed with γH2AX signals generated after etoposide treatment of neurons ( Figure 5 F; p < 0.0001). These results suggest that CTCF-mediated topological structures at the promoters of early-response genes could stimulate the nucleation of Topo IIβ.

We next performed motif scans at Topo IIβ binding sites under basal conditions. Interestingly, these studies revealed that the CTCF transcription factor binding site motif (CTCF_1; http://www.broadinstitute.org/∼pouyak/motifs-table/ ) is the most highly enriched at Topo IIβ binding sites. Under basal conditions, a 37-fold enrichment for this motif at Topo IIβ binding sites was detected relative to a shuffled control version of this binding site found at a similar number of places in the genome (p value = 1 × 10). Similarly, a 31-fold enrichment of the CTCF motif at Topo IIβ peaks was observed under NMDA-treated conditions (p value < < 1 × 10). In fact, in both cases, Topo IIβ disposition at these sites was tightly confined to the CTCF motif itself ( Figure 5 B). CTCF is an architectural protein that creates topological boundaries through chromatin looping and thereby governs interactions between various regulatory regions, such as promoters and enhancers (). A comparison with two different existing CTCF ChIP-seq datasets (GEO: GSM918727 and) revealed that Topo IIβ signals are significantly enriched at CTCF peaks ( Figures 5 C and 5D). Furthermore, immunoprecipitated Topo IIβ under basal conditions was able to co-precipitate CTCF, and this interaction was markedly stimulated following NMDA treatment ( Figure S5 D).

We sought to understand the properties that could underlie the positional specificity of activity-induced DSBs. We found that promoters that incur activity-induced DSBs already contain RNAPII pre-bound at the TSS, as well as a chromatin environment that is highly permissive for gene expression even under basal conditions ( Figures S5 A and S5B). However, pre-incubation of cultured neurons with DRB (5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole), an inhibitor of RNAPII elongation (), prior to their treatment with NMDA had no effect on activity-induced DSB formation ( Figure S5 C), indicating that DSBs are not likely formed as a by-product of torsional stress generated during transcription elongation. Importantly, we noted that although NMDA causes a 5-fold increase in the number of Topo IIβ peaks, activity-induced DSBs occur at loci that already contain Topo IIβ bound under basal conditions and not at sites of nascent Topo IIβ peaks following NMDA treatment ( Figure 5 A).

(F) Aggregate plots of input-normalized CTCF (violet) () and γH2AX (blue) signals within a 4 kb window relative to the TSS and TTS (gray box) of the loci that show increased γH2AX intensity following etoposide treatment.

(E) Aggregate plots of input-normalized CTCF (violet) () and γH2AX (orange) signals within a 4 kb window relative to the transcription start site (TSS) and transcription termination site (TTS) (gray box) of the loci that show increased γH2AX intensity following NMDA treatment.

(D) A similar analysis to (C) was conducted using another publicly available CTCF ChIP-seq dataset from mouse neural progenitors (). Grey bar and lines are as in (C).

(C) Publicly available CTCF ChIP-seq datasets from the cortical plate of 8 week-old mice (GEO: GSM918727 ) were used to determine the overlap of CTCF and Topo IIβ binding profiles at CTCF motifs that were enriched for Topo IIβ peaks in (B). The gray bar in the middle indicates the width of the CTCF peak at these sites. Dashed line denotes the profile of Topo IIβ under basal conditions (control), whereas the solid line indicates Topo IIβ profiles in NMDA-treated samples.

(B) Motif scans at genome-wide Topo IIβ binding sites under basal conditions revealed a strong enrichment for the CTCF transcription factor binding site motif (CTCF_1; http://www.broadinstitute.org/∼pouyak/motifs-table/ ) in the vicinity of Topo IIβ peaks. The plot denotes the disposition of input-normalized Topo IIβ signals relative to CTCF sites that displayed Topo IIβ peaks in their vicinity. Dashed line denotes the profile of Topo IIβ under basal conditions (control), whereas the solid line indicates Topo IIβ profiles in NMDA-treated samples.

(A) Topo IIβ peaks in NMDA-treated samples were categorized into two groups—Class I represents new Topo IIβ peaks that appear after NMDA treatment and Class II denotes Topo IIβ peaks that are present under both basal and NMDA-treated conditions. Aggregate plots of γH2AX signals within a 4 kb window relative to Topo IIβ peaks in each class were then generated as in Figure 4 F.

(E) Box plots indicating the distance of the nearest CTCF peak upstream of the TSS for genes that accumulate γH2AX after treatment with either NMDA or etoposide. CTCF peaks were significantly closer to the TSS of these loci compared to all other genes (p < 0.05, Wilcox test).

(D) Cultured primary neurons were treated with NMDA as before, following which cells were lysed and Topo IIβ was immunoprecipitated. The precipitates were then electrophoresed through 6% SDS-PAGE gels and analyzed by western blotting with the indicated antibodies.

(C) Cultured primary neurons were pre-incubated with DRB (100 μM final) for 15 min, followed by treatment with NMDA as before. Genomic DNA was isolated and amplification of the Fos and β-globin promoter was assessed using qPCR (n = 3, ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA).

(B) IGV genome browser snapshots depicting the disposition of the indicated chromatin marks and transcription factors at the Fos and Npas4 loci under basal conditions.

(A) Publicly available RNAPII ChIP-seq datasets from cultured primary neurons under basal conditions and after stimulation with KCl () were utilized to generate aggregate plots of RNAPII binding profiles at the loci that incur DNA DSBs in response to NMDA treatment of cultured primary neurons. Gray line denotes the RNAPII binding profile under basal conditions whereas the green line represents RNAPII binding following KCl stimulation.

Topo IIβ-Mediated DNA DSBs Facilitate the Expression of Early-Response Genes

Ran et al., 2013 Ran F.A.

Hsu P.D.

Wright J.

Agarwala V.

Scott D.A.

Zhang F. Genome engineering using the CRISPR-Cas9 system. Figure 6 Topo IIβ-Mediated DNA DSBs Govern the Expression of Early-Response Genes Following Neuronal Activity Show full caption (A) Schematic of Fos and Npas4 genes with arrows indicating the positions of sgRNA-directed DNA cleavage by Cas9 (B) HEK293T cells were transfected with luciferase reporter constructs under the control of either Fos or Npas4 upstream sequences, together with the indicated sgRNA and Cas9-carrying constructs and Renilla. Three distinct sgRNAs were used for each locus (#1-#3). Luciferase expression was measured 16 hr after transfection. As a control, luciferase reporter constructs in each case were transfected with Cas9 and sgRNAs directed to the Bdnf promoter (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA) (C) Cultured primary neurons were infected with lentiviral vectors carrying Cas9 and sgRNAs directed to either the Fos or the Npas4 promoter. RNA was extracted 8 days post-infection and the expression of Fos and Npas4 was determined relative to neurons infected with sgRNAs directed to the Bdnf promoter using qRT-PCR (n = 3, ∗∗p < 0.01, two-tailed t test). ∗p < 0.05, two-way ANOVA). (D) Cultured primary neurons were treated with NMDA as before and allowed to recover in NMDA-free media. Topo IIβ ChIP was then performed at the indicated times and the amplification of the Fos promoter was assessed as in Figure 3 A (n = 3,p < 0.05, two-way ANOVA). (E) Cultured primary neurons were treated with NMDA as before and allowed to recover in NMDA-free media. RNA was then extracted at the indicated times and the expression of Fos, Npas4, and Egr1 was assessed using qRT-PCR (n = 4, ∗∗∗p < 0.001, one way-ANOVA). (F) Cultured primary neurons were incubated with a specific inhibitor of DNA-PK (NU7026) for 1 hr, following which neurons were treated with NMDA and allowed to recover in NMDA-free media. RNA was extracted 2 hr after the initial NMDA treatment (time = 0, x axis), and the levels of Fos, Npas4, and Egr1 was assessed relative to neurons treated with NMDA in the absence of NU7026 using qRT-PCR (n = 4, ∗p < 0.05, two-tailed t test). (G) Cultured primary neurons were infected with lentiviral vectors carrying either a scrambled shRNA (control) or one of two distinct shRNAs against Top2b (shRNA#1 or shRNA#2). One week after the infection, neurons were treated with NMDA (50 μM) for 10 min followed by recovery in NMDA-free media for an additional 10 min. Enrichment of γH2AX within exons of Fos, Npas4, and Egr1 was assessed using ChIP. As a control, the Fos promoter region was also probed (n = 3, ∗∗p < 0.01, two-way ANOVA). (H) Cultured primary neurons were infected with Top2b shRNAs and treated with NMDA as in (G). RNA was then extracted and the levels of Fos and Npas4 were probed using qRT-PCR (n = 4, ∗∗p < 0.01, one-way ANOVA). (I) Scrambled and Top2b shRNAs were stereotactically injected into the hippocampus of two-month old C57BL/6 mice. Four weeks after the injections, acute hippocampal slices were prepared and LTP was induced by 3× TBS at the Schaffer collateral-CA1 synapses. Sample traces represent fEPSPs 1 min before (gray) and 1 hr after (black) TBS. Scale bars, 1 mV and 20 ms (5–6 slices per animal, 3 animals per group, ∗p < 0.05, one-way ANOVA). (J) Cultured primary neurons were infected with a combination of lentiviral vectors carrying Cas9 and sgRNAs directed to the Fos promoter (CRISPR) together with Top2b shRNAs. Controls represent neurons infected with Cas9 and sgRNAs directed to the Bdnf promoter together with scrambled shRNAs (control). One week after the lentiviral infections, neurons were treated with NMDA (50 μM) for 10 min followed by recovery in NMDA-free media for 10 min. RNA was then extracted and Fos expression was assessed using qRT-PCR (n = 3, ∗p < 0.05 one-way ANOVA). Because DSB formation within the promoters of early-response gene correlates with their expression, we tested whether DSBs have an effect on the expression of these genes. To begin, we obtained luciferase reporter constructs that were under the control of either the Fos or the Npas4 regulatory regions, and utilized the CRISPR-Cas9 system to generate targeted DSBs within the promoters of Fos and Npas4 () ( Figure 6 A). Transfection of matching luciferase reporter and Cas9 constructs in HEK293T cells caused a marked increase in luciferase expression in both cases compared to controls in which Cas9 was targeted to cleave the Bdnf promoter ( Figure 6 B). An upregulation of endogenous Fos and Npas4 was also detected when cultured primary neurons were infected with lentiviral vectors carrying Cas9 and the appropriate sgRNAs ( Figure 6 C). These results suggest that the formation of DSBs within the promoters of early-response genes stimulates their expression.

Figure S6 Effects of Top2b Knockdown on Arc Expression and Basal Synaptic Transmission Show full caption (A) Cultured primary neurons were incubated either in the presence or absence of NU7026 (DNA-PKi) and treated with NMDA as before. Neurons were allowed to recover in NMDA-free media for 2 hr. Genomic DNA was isolated and amplification of the Fos and β-globin promoter was assessed using qPCR (n = 3, ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA). (B) Cultured primary neurons were infected with lentiviral vectors carrying either a scrambled shRNA or one of two distinct shRNAs against Top2b (shRNA#1 and shRNA#2, respectively). One week after infection, RNA was extracted and Top2b expression was assessed using qRT-PCR (n = 4, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA). (C) Cultured primary neurons were infected with Top2b shRNAs and treated with NMDA as in Figure 6 G. RNA was then extracted and the levels of Arc were probed using qRT-PCR. (D) Scrambled and Top2b shRNAs were stereotactically injected into the hippocampus of two-month old C57BL/6 mice. Four weeks after the injections, acute hippocampal slices were prepared and synaptic input-output relationship was obtained by plotting the slopes of evoked fEPSPs against fiber-volley amplitude (5-6 slices per animal, 3 animals per group). To further understand how the presence of activity-induced DSBs affects early-response gene expression, we assessed DNA repair kinetics of activity-induced DSBs. Because DSB formation within the Fos promoter precludes the amplification of this region in PCR assays ( Figure 3 F), the recovery of amplification was used to indicate successful repair at this locus. Topo IIβ ChIP-qPCR at various times after NMDA treatment of cultured primary neurons indicated a significant reduction in PCR amplification of the Fos promoter region at 30 min after the initial NMDA stimulus ( Figure 6 D). However, PCR amplification was restored by 2 hr after NMDA stimulation ( Figure 6 D). Similar results were also observed in PCR assays with genomic DNA directly isolated from cultured primary neurons following NMDA treatment ( Figure S6 A). These results suggest that activity-induced DSBs are repaired within 2 hr of the initial stimulus. Interestingly, Fos, Npas4, and Egr1 mRNA levels after NMDA treatment followed similar dynamics, with transcript levels being markedly upregulated at 30 min after the initial stimulus, but retuning to basal levels by 2 hr after stimulation ( Figure 6 E). Thus, the expression patterns of early-response genes correlate well with the formation and repair of activity-induced DSBs.

Veuger et al., 2003 Veuger S.J.

Curtin N.J.

Richardson C.J.

Smith G.C.

Durkacz B.W. Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. We next tested the effects of perturbing DSB repair on the expression of early-response genes by pre-incubating neurons with a specific inhibitor of DNA-PK (NU7026), which is an essential component of DSB repair through nonhomologous end joining (NHEJ) (). Pre-incubation with NU7026 prevented the PCR amplification of the Fos promoter regions even after recovery for 2 hr following NMDA treatment, indicating that the repair of activity-induced DSBs is dependent on NHEJ ( Figure S6 A). Like before ( Figure 6 E), Fos, Npas4, and Egr1 expression was markedly upregulated upon stimulation and returned to baseline levels by 2 hr post-stimulation in untreated controls. Incubation with NU7026 had no effect on either the basal expression of early-response genes or on their peak induction levels post-stimulation (data not shown). However, early-response genes in NU7026-treated samples were delayed in returning to basal levels and were upregulated at 2 hr post-stimulation relative to untreated controls ( Figure 6 F), although the expression of these genes at 2 hr was still lower than their peak induction levels at 30 min post-stimulation (data not shown). These results suggest that the repair of activity-induced DSBs can affect the dynamics of early-response gene expression.

To clarify the role of Topo IIβ in activity-induced DSB formation and early-response gene expression, we infected cultured primary neurons with lentiviral vectors carrying shRNAs against Top2b. qRT-PCR experiments performed one week after the lentiviral infections using two distinct shRNAs revealed that both shRNAs were able to knockdown Top2b expression by at least 50% ( Figure S6 B). To examine the effects of Top2b knockdown on DSB formation, we assessed γH2AX enrichment within the exons of early-response genes after NMDA treatment using ChIP-qPCR. Whereas NMDA treatment caused a robust increase in γH2AX levels in the exons of Fos, Npas4, and Egr1 in neurons infected with a scrambled shRNA, this increase was attenuated in neurons infected with Top2b shRNAs ( Figure 6 G). In addition to this, qRT-PCR experiments following NMDA treatment of cultured primary neurons revealed that whereas neurons infected with scrambled shRNA showed a significant induction of Fos and Npas4 transcripts following activity stimulation, the induction of these genes was severely attenuated in neurons infected with Top2b shRNAs ( Figure 6 H). In contrast to Fos and Npas4, the expression of another early-response gene, Arc, which does not incur activity-induced DNA DSBs, was not affected by Top2b knockdown ( Figure S6 C). These results suggest that Topo IIβ is essential for activity-induced DSB formation, as well as for the expression of genes that incur activity-induced DSBs.

To evaluate how the loss of Topo IIβ affects synaptic functions, we stereotactically injected lentiviral vectors carrying Top2b shRNAs into the CA1 region of the mouse hippocampus. Four weeks after viral injections, we performed extracellular recordings in acute hippocampal slices. As assessment of basal synaptic transmission revealed no significant differences between control slices infected with a scrambled shRNA and slices infected with Top2b shRNAs ( Figure S6 D). However, theta-burst stimulation (TBS)-induced LTP from Schaffer collateral-CA1 synapses was severely impaired in slices infected with Top2b shRNAs ( Figure 6 I). While the slices transduced with scrambled shRNA exhibited 200% fEPSP over baseline, shRNA#1 transduced slices showed 130% and shRNA#2 transduced slices showed 150% fEPSP over baseline over the time course of 60 min following theta-burst stimulation ( Figure 6 I). These results are consistent with a role for Topo IIβ in the expression of early-response genes and maintaining synaptic function.