Article Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes
Graphical Abstract
Highlights
d Neuronal activity causes th
e formation o
f DNA double strand
breaks(DSBs)
d Activity-induced DSBs form within th
e promoters o
f a subset
of early-response genes
d Topoisomeras
e II b is necessary for activity-induced DSB
formation
d Activity-induced DSBs facilitat
e the expression o
f early-
response genes in neurons Authors
Ram Madabhushi,Fan Gao,
Andreas R.Pfenning,...,Sukhee Cho, Manolis Kellis,Li-Huei Tsai
Correspondence
lhtsai@https://www.doczj.com/doc/e517757760.html,
In Brief
The formation of activity-induced DNA double strand breaks within promoter regions is required for transcription of a subset of neuronal early-response genes that are crucial for experience-driven synaptic changes associated with learning and memory.
Accession Numbers
GSE61887 Madabhushi et al.,2015,Cell161,1592–1605
June18,2015a2015Elsevier Inc.
https://www.doczj.com/doc/e517757760.html,/10.1016/j.cell.2015.05.032
Article Activity-Induced DNA Breaks Govern the Expression
of Neuronal Early-Response Genes
Ram Madabhushi,1Fan Gao,1Andreas R.Pfenning,2,3Ling Pan,1Satoko Yamakawa,1Jinsoo Seo,1Richard Rueda,1 Trongha X.Phan,1Hidekuni Yamakawa,1Ping-Chieh Pao,1Ryan T.Stott,1Elizabeta Gjoneska,1,3Alexi Nott,1
Sukhee Cho,1Manolis Kellis,2,3and Li-Huei Tsai1,3,*
1Picower Institute for Learning and Memory,Department of Brain and Cognitive Sciences
2Computer Science and Arti?cial Intelligence Laboratory
Massachusetts Institute of Technology,Cambridge,MA02139,USA
3The Broad Institute of Harvard and MIT,Cambridge,MA02139,USA
*Correspondence:lhtsai@https://www.doczj.com/doc/e517757760.html,
https://www.doczj.com/doc/e517757760.html,/10.1016/j.cell.2015.05.032
SUMMARY
Neuronal activity causes the rapid expression of im-mediate early genes that are crucial for experience-driven changes to synapses,learning,and memory. Here,using both molecular and genome-wide next-generation sequencing methods,we report that neu-ronal activity stimulation triggers the formation of DNA double strand breaks(DSBs)in the promoters of a subset of early-response genes,including Fos, Npas4,and Egr1.Generation of targeted DNA DSBs within Fos and Npas4promoters is suf?cient to induce their expression even in the absence of an external stimulus.Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase II b(Topo II b),and knockdown of Topo II b attenuates both DSB formation and early-response gene expression following neuronal stimu-lation.Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.
INTRODUCTION
Neurons are endowed with the remarkable ability to sense and process changes in an organism’s external environment.The exposure to a new sensory experience,for instance,profoundly alters the morphology and connectivity of neural circuits,and these changes are thought to be instrumental in the formation of long-lasting memories and adaptive responses(Goelet et al.,1986).The signaling pathways that underlie these experi-ence-dependent changes have been studied extensively and have led to the current orthodoxy that the initiation of new gene transcription programs is crucial for synaptic plasticity. Neuronal activity-regulated genes are classi?ed into different subgroups based on the latency of their expression following an activity-dependent stimulus.Genes induced in the earliest wave,referred to as early-response genes,are enriched for tran-scription factors,such as Fos,Npas4,Egr1,and Nr4a1,and their expression occurs independently of de novo protein synthesis (West and Greenberg,2011).These transcription factors then govern the expression of late-response genes,such as Bdnf, Homer1,Nrn1,and Rgs2,which are induced with relatively slower kinetics.In this way,early-response genes ultimately regulate various synaptogenic processes,including neurite outgrowth,synapse development and maturation,and the bal-ance between excitatory and inhibitory synapses(West and Greenberg,2011).
The de?ning characteristic of early-response genes is their rapid and robust expression within minutes after stimulation.A number of molecular features that facilitate this rapid response have been described previously.For instance,even in the absence of an activity-dependent stimulus,the Fos promoter is already bound by the RNA polymerase II(RNAPII)complex and by activity-dependent transcription factors,such as CREB and SRF.Furthermore,nucleosomes at the Fos promoter already carry chromatin modi?cations that are permissive for active transcription,including histone H3trimethylated at lysine4 (H3K4me3)(Kim et al.,2010).Neuronal activity then variously causes the phosphorylation of CREB and the SRF cofactor, ELK1,the recruitment of the histone acetyltransferase,CBP,to the Fos promoter,and CBP and RNAPII recruitment to enhancer elements of activity-regulated genes(West and Greenberg, 2011).Together,these changes orchestrate the expression of neuronal activity-regulated genes.
Despite these details however,the precise nature of the mo-lecular switch that precludes early-response gene expression under basal conditions,as well as the mechanisms that override this impediment in response to neuronal activity still remain poorly understood.Our?ndings suggest that the expression of early-response genes is subdued by the imposition of topologi-cal constraints and that the rapid resolution of these constraints in response to neuronal activity involves the generation of DNA double strand breaks(DSBs)within their promoters. RESULTS
Etoposide-Induced DSBs Stimulate the Expression of Early-Response Genes
Our results stemmed from some unexpected observations made while studying the effects of DSB formation in neurons.
The
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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 (Dobbin et al.,2013;Kim et al.,2008;Madabhushi et al.,2014;Wang et al.,2013).
With the idea of further characterizing the consequences of DSB formation in neurons,we incubated cultured primary neurons with etoposide for 6hr and performed gene expression pro?ling 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 692genes that were differentially expressed compared to vehicle-treated controls.Consistent with the ex-pectation that DSBs would interfere with transcription,an over-whelming majority (680genes)of the differentially expressed genes were downregulated (Figure 1A and Table S1).Remark-ably,however,the 12genes that were upregulated were en-riched for neuronal activity-regulated genes and particularly the so-called early-response genes,such as Fos ,FosB ,
and
A B C
D E F
Figure 1.Early-Response Genes Are Upregulated following Etoposide Treatment of Neurons
(A)Cultured primary neurons (DIV 10)were incubated with either vehicle (DMSO)or etoposide (5m M)for 6hr,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 signi?cantly (p <0.05)are indicated in red.(Bottom)Schematic indicating the number of downregulated (blue)and upregulated genes (red).
(B)List of upregulated genes in etoposide-treated neurons and their relative fold change (log 2)compared to vehicle-treated controls.
(C)UCSC genome browser snapshots of RNA-seq trace ?les 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.
(D)Cultured primary neurons were treated with either etoposide (5m M)or vehicle (DMSO)for 20min,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).
(E)Cultured primary neurons were pre-incubated with the ATM inhibitor,KU55933(ATMi),for 30min following which etoposide treatment was performed as in (D).(Top)Neurons were immunostained with antibodies against g H2AX 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).
(F)Cultured primary neurons were incubated with the indicated drugs for 20min,following which the expression of Fos and Npas4was assessed using qRT-PCR (n =3,**p <0.01,one-way ANOVA).
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Npas4—transcription factors that are also rapidly expressed in response to neuronal activity(Figures1B and1C).
Based on these observations,we directly assessed the ex-pression of various neuronal activity-regulated genes after eto-poside treatment of cultured primary neurons using quantitative real-time PCR(qRT-PCR).Early-response genes,including Fos, FosB,Npas4,and Egr1were all upregulated within20min of treatment with etoposide(Figure1D).However,other activity-regulated genes,such as Bdnf and Homer1,showed no such increase in either the RNA-seq or the qRT-PCR experiments (Figures1C and1D).Thus,etoposide selectively induces a small subset of activity-regulated genes.
We then evaluated whether etoposide-mediated early-res-ponse gene expression is a result of the activation of the DNA damage response.Cultured primary neurons pre-incu-bated with a speci?c inhibitor(KU55933;ATMi)against ATM (ataxia telangiectasia mutated)caused a marked reduction in DSB signaling,as indicated by a reduction in the intensity of g H2AX,a marker of DSB signaling(Figure1E).However,ATM inhibition had no effect on the etoposide-mediated increase in Fos and Npas4expression(Figure1E).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,neo-carzinostatin(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(Figure1F).Together,our results unexpectedly re-vealed that Topo II-mediated DSBs stimulate the expression of early-response genes.
Neuronal Activity Results in the Formation of DNA DSBs Because early-response genes are normally induced in res-ponse to stimulation of neuronal activity,we tested whether established paradigms of neuronal stimulation are also associ-ated 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 Npas4mRNA(Figures S1A and S1B).Interestingly, each of these treatments also caused an increase in the levels of g H2AX,an established marker of DNA DSBs(Figure S1C) (Crowe et al.,2006).
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 g H2AX levels compared to untreated controls(Figures S1D and S1E).To understand whether DSBs are also formed following neuronal activity in vivo,we subjected wild-type C57BL/6mice to a training paradigm for contextual fear conditioning,following which we prepared hippocampal lysates and measured g H2AX levels.Similar to our observations with cultured primary neurons and hippocampal slices,elevated g H2AX levels were detectable in hippocampal lysates within 15min after exposure to the fear-conditioning paradigm(Fig-ure S1F).Moreover,DSB formation has also been reported in other neuronal stimulation paradigms(Suberbielle et al., 2013).Thus,neuronal activity correlates with the formation of DNA DSBs in neurons.Neuronal Activity-Induced DNA DSBs Form at Speci?c Locations in the Genome
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 Ser139in the vicinity of DSB sites(Rogakou et al.,1998).The identi?cation of chromatin enriched for g H2AX can be exploited to derive the locations of DSBs(Iacovoni et al.,2010;Rodriguez et al.,2012).We therefore stimulated cultured primary neurons by brie?y incubating them with50m M NMDA and then performed genome-wide g H2AX ChIP-seq.
For an initial assessment of genome-wide g H2AX ChIP-seq signals,we classi?ed genomic regions into14distinct chro-matin 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 S2A)(Ernst et al.,2011;Gjoneska et al.,2015).Corre-lating the positional information of these chromatin marks with raw g H2AX ChIP-seq signals revealed that the observed in-crease in g H2AX is con?ned largely to actively transcribed genes and their downstream regions,but not to enhancers,polycomb repressed regions,or heterochromatin(Figure2A).
To further characterize the distribution of enriched g H2AX signals within genomic regions,we performed differential peak calling(Experimental Procedures)and observed that g H2AX enrichment following NMDA treatment occurs primarily within gene bodies compared to distal intergenic regions(Figure2B). Surprisingly,our analysis revealed only21regions that were enriched for g H2AX signals in the NMDA-treated samples compared to controls.Twenty of these regions were within genes, whereas one site was detected in intergenic regions(Figure2C). Remarkably,included within these21loci were the early-response genes,Fos,FosB,Npas4,Egr1,Nr4a1,and Nr4a3(Fig-ure2C).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 g H2AX(Figure2C).Addition-ally,the loci identi?ed by differential peak calling also displayed the highest g H2AX intensities in the NMDA-treated samples,but not in vehicle-treated samples(Tables S2and S3).
A closer examination of g H2AX distribution at these loci, including at the early-response genes,revealed a peculiar pattern,with g H2AX peaks initiating adjacent to the transcrip-tional start site(TSS),spreading into the gene body,and termi-nating downstream of the30UTR(Figure2D and Figure S2B).In fact,g H2AX peak width was strictly proportional to gene length (Figure S2C).The only exception to this scenario was the case of Homer1,in which g H2AX signals only spanned across the anno-tated short isoform of Homer1(Figure S2B).While the signi?-cance of this is presently unclear,it is noteworthy that only the short isoform of Homer1is regulated by neuronal activity(Sala et al.,2003).In contrast to these early-response genes,none of the other late response genes,including Bdnf,Rgs2,Nrn1, and Gpr3showed an increase in g H2AX intensity in their vicinity following NMDA treatment(Figure2D and Figure S2B).Together
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these results indicate that activity-dependent stimulation of neu-rons results in the formation of DNA DSBs at very speci?c loca-tions in the genome,particularly near early-response genes.Neuronal Activity-Induced DNA DSBs Are Generated by Topoisomerase II b
In an effort to determine the mechanisms that underlie the forma-tion of neuronal activity-induced DSBs,we returned to our orig-inal observation that etoposide treatment is speci?cally able to upregulate the expression of early-response genes (Figure 1F).Etoposide introduces DSBs by targeting Topo II (Vos et al.,2011).Mammalian cells express two distinct isoforms of Topo II,Topo II a ,and Topo II b .Topo II a is mainly expressed in dividing cells,whereas Topo II b is robustly expressed in postmitotic cells,including neurons,and is primarily implicated in transcription-related functions (Austin and Marsh,1998).Incidentally,Topo II b -mediated DSBs were previously shown to be essential for estradiol-stimulated activation of gene expression (Ju et al.,2006).These observations caused us to focus speci?cally on Topo II b .
We began by assessing Topo II b binding at various regions of the prototypical early-response gene,Fos ,under
basal
A B
C D
Figure 2.Neuronal Activity-Induced DNA DSBs Form at Early-Response Genes
(A)Genomic regions were categorized into 14distinct chromatin states based on combinatorial patterns of various chromatin marks (Figure S2A).The percentage of g H2AX peaks within each chromatin state were then normalized to the proportion of the genome with that chromatin state and plotted.
(B)g H2AX ChIP-seq signals enriched in NMDA-treated samples relative to controls were processed using CEAS (Cis-Regulatory Element Annotation System)program (https://www.doczj.com/doc/e517757760.html,/CEAS/).(Left)Pie chart depicting the relative proportions of the indicated annotated regions in the genome.(Right)Disposition of g H2AX signals within these annotated genomic regions.
(C)Differential peak calling was performed to determine the regions that were enriched for g H2AX following NMDA treatment (Experimental Procedures ).This data were then processed using CIRCOS software (Krzywinski et al.,2009)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 g H2AX signals.Red lines within the green ring represent loci that were enriched for g H2AX relative to controls.Twenty loci were within genes,and these genes are indicated.One locus was within intergenic regions.
(D)UCSC genome browser views depicting the disposition of g H2AX 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.
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conditions.Topo II b ChIP-qPCR indicated negligible binding in the exons of the Fos gene,as well as within the promoters of b -globin and GAPDH (Figure 3A).However,Topo II b binding was signi?cantly enriched within the Fos promoter (Figure 3A).Similarly,Topo II b binding was also enriched within the Npas4promoter (Figure 3A).These results suggest that Topo II b is bound to the promoters of early-response genes under basal conditions.
The regulatory elements that bind the Fos promoter are well characterized,and additional ChIP experiments revealed that Topo II b co-occupies the Fos promoter together with ELK1,as well as the lysine deacetylase,HDAC2(Figure 3B and Fig-ure S3A).We previously showed that HDAC2binds to the promoters of several early-response genes and negatively mod-ulates their expression (Guan et al.,2009).While screening for proteins that could potentially regulate HDAC2binding to
these
A B C D
E F G
Figure 3.Topo II b Binds to the Promoters of Early-Response Genes under Basal Conditions and Cleaves Them in Response to Neuronal Activity
(A)ChIP analysis of Topo II b binding to the promoters of Fos and Npas4.Two distinct primer sets (Fos Prom#1and Fos Prom#2,respectively)were used to probe the Fos promoter region.In addition,two different exons within the Fos gene (exons 2and 4),as well as the promoters of Npas4,b -globin ,and GAPDH ,were also probed (n =3,*p <0.05,**p <0.01,one-way ANOVA).
(B)Sequential ChIP analysis of HDAC2and Topo II b binding to the Fos promoter.Cultured primary neurons were ?rst subjected to ChIP with antibodies against Topo II b .The crosslinked proteins were then immunoprecipitated with antibodies against HDAC2.Primers were as in (A)(n =3,*p <0.05,one-way ANOVA).(C)Topo II b was immunoprecipitated from cultured primary neurons following NMDA treatment.The precipitated Topo II b was then incubated with 1m g of a supercoiled luciferase reporter plasmid carrying $6kb of upstream regions of the Npas4gene.Reactions were then incubated at 30 C for 15min,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).
(D)Luciferase reporter constructs containing sequences upstream of either the Fos TSS or the Npas4TSS were incubated with puri?ed recombinant human Topo II b (8units/reaction)either in the presence or absence of etoposide (0.2mM ?nal).Reactions were then incubated at 30 C for 15min,stopped,and electro-phoresed through 1%agarose gels.As controls,constructs lacking the Fos and Npas4sequences (D Fos-luc and D Npas4-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.
(E)Schematic showing how DNA cleavage by Topo II b (red ovals)would preclude the ampli?cation of the Fos promoter by PCR primers utilized in (A)(indicated by blue and green arrows).
(F)ChIP analysis of Topo II b 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).
(G)ChIP analysis of ELK1binding at the indicated regions under basal conditions and following NMDA treatment (n =3,**p <0.01,two-way ANOVA).
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promoters,we separately discovered that the protein,tyrosyl DNA phosphodiesterase2(TDP2),binds HDAC2and is enriched at several early-response gene promoters,including Fos and Npas4,under basal conditions(Figure S3B and data not shown). These results are intriguing because Topo II-mediated DSBs involve the formation of a covalent phosphotyrosyl bond be-tween Topo II and the DNA,and TDP2specializes in processing these intermediates and in the repair of Topo II-mediated DSBs (Cortes Ledesma et al.,2009).
To test whether Topo II b activity could underlie activity-induced DSB formation,we?rst measured levels of Topo II b cleavage complexes following NMDA treatment using an im-mune complex of enzyme(ICE)assay(Nitiss et al.,2012).As a control,we incubated neurons with etoposide and observed an increase in Topo II b covalent complexes(Figure S3C).Interest-ingly,NMDA treatment also caused an increase in Topo II b cova-lent complexes,indicating that neuronal activity results in Topo II b-mediated DNA cleavage(Figure S3C).To further assess how neuronal activity affects Topo II b,we immunoprecipitated Topo II b following NMDA treatment of primary neurons and incu-bated the precipitated Topo II b with supercoiled plasmids.Incu-bation with Topo II b caused a potent relaxation of supercoiled plasmids in both NMDA-treated and vehicle-treated samples (Figure3C).However,plasmids incubated with Topo II b from NMDA-treated samples also displayed increased smearing, indicative of fragmented DNA(Figure3C).These results suggest neuronal activity confers Topo II b with an increased propensity to generate DNA breaks.
We next performed DNA cleavage assays in which we incu-bated puri?ed,recombinant Topo II b together with supercoiled plasmids that either contained or lacked the upstream se-quences of the Fos gene.As expected,incubation of supercoiled plasmids with Topo II b caused an increase in relaxed(Form II) DNA(Figure3D).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(Figure3D).Similar results were also observed with plasmids containing Npas4upstream sequences.Together, these results suggest that Topo II b preferentially cleaves the promoters of early-response genes.
Based on Topo II b binding in the promoters of early-response genes,we then speculated that if Topo II b generates DSBs in these promoters in an activity-dependent manner,that such cleavage should prevent the ampli?cation of these regions using the primers that detect Topo II b enrichment(Figure3E).Control Topo II b ChIP experiments following etoposide treatment of cultured primary neurons revealed a signi?cant reduction in ampli?cation by two distinct primer sets that span the Fos pro-moter(Figure3F).Importantly,Topo II b ChIP following NMDA treatment also indicated a sharp reduction in the ampli?cation of the Fos promoter regions(Figures3F and S3D).A similar attenuation in PCR ampli?cation was also observed following ELK1ChIP in NMDA-treated neurons(Figure3G).To determine whether activity-induced DNA cleavage occurs at a speci?c site within the Fos promoter,we repeated the PCR ampli?cation assays after Topo II b ChIP using various primer combinations that spanned the originally identi?ed Topo II b binding region within the Fos promoter.However,each region that showed enriched ampli?cation following Topo II b ChIP under basal con-ditions also showed reduced ampli?cation following NMDA treatment(Figure S3E).Thus,activity-induced DSBs are not site-speci?c 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 II b.
Genome-wide Topo II b Cleavage Patterns Coincide with Sites of Activity-Induced DNA DSBs
To further examine the connections between Topo II b and activ-ity-induced DSBs,we performed Topo II b ChIP-seq under basal conditions and following neuronal activity.An analysis of Topo II b signals within various genomic regions suggested that under basal conditions,Topo II b 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(Figure4A and Table S4).A comparison with existing datasets of various factors that bind activity-regulated genes(Kim et al.,2010)revealed that Topo II b binding is enriched at SRF and CREB binding sites,as well as CBP binding sites within promoters and enhancers(Figures4B and4C).These re-sults further indicate that Topo II b binding patterns overlap with regulators of neuronal activity-induced gene expression. Interestingly,NMDA caused a substantial increase in genome-wide Topo II b binding.Whereas430Topo II b peaks were identi-?ed under basal conditions,2,416peaks were detected within 20min of the initial NMDA treatment(Table S4).This nearly 5-fold increase in Topo II b signals was largely proportionally distributed within the same chromatin regions that showed Topo II b binding under basal conditions(Figure4A and Table S4).NMDA treatment also caused an enrichment of Topo II b sig-nals at SRF,CREB,and CBP binding sites(Figures4B and4C). These features also extend to the promoters of early-response genes,such as Fos and Npas4,where an increase in Topo II b sig-nals were clearly detected following neuronal activity(Figure4D). Importantly,Topo II b signals under both basal and NMDA-treated conditions were found immediately adjoining g H2AX-en-riched regions in NMDA-treated samples(Figure4D).These results further suggest that Topo II b binding patterns are consis-tent with their role in activity-dependent DNA DSB formation. To further understand whether Topo II b-mediated DNA cleav-age could underlie the selective pattern of DSB formation in response to neuronal activity,we performed g H2AX ChIP-seq after treatment of cultured primary neurons with etoposide. Similar to results with NMDA treatment,g H2AX signals after eto-poside treatment were enriched primarily within gene bodies compared to intergenic regions(Figure4E).Furthermore,aggre-gate plots revealed that etoposide-induced and NMDA-induced g H2AX signals display a strikingly similar distribution pattern at the sites of activity-induced DSBs(Figures4F and S4A),and this similarity is further emphasized from the comparison of NMDA and etoposide-induced g H2AX signals at individual genes(Figure S4B).These data suggest that Topo II b-mediated DNA cleavage could underlie activity-induced DSB formation in neurons.
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A B
C D
E F
Figure4.Genome-wide Topo II b DNA Cleavage Patterns Coincide with the Sites of Neuronal Activity-Induced DSBs
(A)Genomic regions were categorized into14distinct chromatin states based on combinatorial patterns of various chromatin marks(Figure S2A).The percentage of Topo II b peaks within each chromatin state were then normalized to the proportion of the genome within that chromatin state and plotted.
(B)Publicly available ChIP-seq datasets of SRF and CREB(Kim et al.,2010)were used to determine the binding pro?les of Topo II b relative to the binding sites of these proteins.The graphs indicate the averaged binding patterns of Topo II b within a4kb window of all SRF(left)and CREB(right)peaks.Dashed line indicates the pro?le under basal conditions,whereas the solid line depicts the pro?les following NMDA treatment.
(C)Binding pro?les of Topo II b within a4kb window of CBP peaks at either promoters(left)or enhancers(right)were generated as in(B).
(D)UCSC genome browser views denoting the disposition of g H2AX and Topo II b signals at Fos and Npas4under the indicated conditions.y axis denotes signal intensity and the range is indicated in parentheses.
(E)g H2AX ChIP-seq signals enriched in etoposide-treated samples relative to controls were processed using CEAS(Cis-Regulatory Element Annotation System) program(https://www.doczj.com/doc/e517757760.html,/CEAS/).(Left)Pie chart depicting the relative proportions of the indicated annotated regions in the genome as in Figure2A. (Right)Disposition of differential g H2AX signals within these annotated genomic regions following etoposide treatment.
(F)Aggregate plots of input-normalized g H2AX signals at the20loci that show increased g H2AX 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 a2kb 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.
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Activity-Induced DNA DSBs Occur within
CTCF-Generated Topological Domains
We sought to understand the properties that could underlie the positional speci?city 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 environ-ment that is highly permissive for gene expression even under basal conditions(Figures S5A and S5B).However,pre-incuba-tion of cultured neurons with DRB(5,6-Dichloro-1-b-D-ribofura-nosylbenzimidazole),an inhibitor of RNAPII elongation(Yankulov et al.,1995),prior to their treatment with NMDA had no effect on activity-induced DSB formation(Figure S5C),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 a5-fold increase in the number of Topo II b peaks,activity-induced DSBs occur at loci that already contain Topo II b bound under basal conditions and not at sites of nascent Topo II b peaks following NMDA treatment (Figure5A).
We next performed motif scans at Topo II b binding sites under basal conditions.Interestingly,these studies revealed that the CTCF transcription factor binding site motif(CTCF_1;http:// https://www.doczj.com/doc/e517757760.html,/$pouyak/motifs-table/)is the most highly enriched at Topo II b binding sites.Under basal conditions, a37-fold enrichment for this motif at Topo II b binding sites was detected relative to a shuf?ed control version of this binding site found at a similar number of places in the genome(p value=13 10à70).Similarly,a31-fold enrichment of the CTCF motif at Topo II b peaks was observed under NMDA-treated conditions (p value<<1310à100).In fact,in both cases,Topo II b disposition at these sites was tightly con?ned to the CTCF motif itself(Fig-ure5B).CTCF is an architectural protein that creates topological boundaries through chromatin looping and thereby governs inter-actions between various regulatory regions,such as promoters and enhancers(Ong and Corces,2014).A comparison with two different existing CTCF ChIP-seq datasets(GEO:GSM918727 and Phillips-Cremins et al.,2013)revealed that Topo II b signals are signi?cantly enriched at CTCF peaks(Figures5C and5D). Furthermore,immunoprecipitated Topo II b under basal condi-tions was able to co-precipitate CTCF,and this interaction was markedly stimulated following NMDA treatment(Figure S5D). To determine whether the association between Topo II b 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 g H2AX signals at the sites of activity-dependent DNA DSBs.Our analysis revealed that the probability of?nding a CTCF peak within2kb of the bound-ary of g H2AX regions was signi?cantly greater than the prob-ability of?nding permutated random sites in the mappable re-gions throughout the genome(p<0.0001).Furthermore,CTCF peaks lie signi?cantly closer to the TSS of genes that incur DSBs following NMDA treatment compared to other genes(Fig-ure S5E).Aggregate plots of CTCF and g H2AX distribution at sites of activity-induced DSBs further revealed that CTCF peaks tightly envelop g H2AX regions within these loci(Figure5E). Similar results were also observed with g H2AX signals generated after etoposide treatment of neurons(Figure5F;p<0.0001). These results suggest that CTCF-mediated topological struc-tures at the promoters of early-response genes could stimulate the nucleation of Topo II b.
Topo II b-Mediated DNA DSBs Facilitate the Expression of Early-Response Genes
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 con-trol of either the Fos or the Npas4regulatory regions,and utilized the CRISPR-Cas9system to generate targeted DSBs within the promoters of Fos and Npas4(Ran et al.,2013)(Figure6A).Trans-fection of matching luciferase reporter and Cas9constructs in HEK293T cells caused a marked increase in luciferase expres-sion in both cases compared to controls in which Cas9was tar-geted to cleave the Bdnf promoter(Figure6B).An upregulation of endogenous Fos and Npas4was also detected when cultured primary neurons were infected with lentiviral vectors carrying Cas9and the appropriate sgRNAs(Figure6C).These results suggest that the formation of DSBs within the promoters of early-response genes stimulates their expression.
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 for-mation within the Fos promoter precludes the ampli?cation of this region in PCR assays(Figure3F),the recovery of ampli?ca-tion was used to indicate successful repair at this locus.Topo II b ChIP-qPCR at various times after NMDA treatment of cultured primary neurons indicated a signi?cant reduction in PCR ampli-?cation of the Fos promoter region at30min after the initial NMDA stimulus(Figure6D).However,PCR ampli?cation was restored by2hr after NMDA stimulation(Figure6D).Similar re-sults were also observed in PCR assays with genomic DNA directly isolated from cultured primary neurons following NMDA treatment(Figure S6A).These results suggest that activ-ity-induced DSBs are repaired within2hr of the initial stimulus. Interestingly,Fos,Npas4,and Egr1mRNA levels after NMDA treatment followed similar dynamics,with transcript levels being markedly upregulated at30min after the initial stimulus,but re-tuning to basal levels by2hr after stimulation(Figure6E).Thus, the expression patterns of early-response genes correlate well with the formation and repair of activity-induced DSBs.
We next tested the effects of perturbing DSB repair on the expression of early-response genes by pre-incubating neurons with a speci?c inhibitor of DNA-PK(NU7026),which is an essential component of DSB repair through nonhomologous end joining (NHEJ)(Veuger et al.,2003).Pre-incubation with NU7026pre-vented the PCR ampli?cation of the Fos promoter regions even after recovery for2hr following NMDA treatment,indicating that the repair of activity-induced DSBs is dependent on NHEJ(Fig-ure S6A).Like before(Figure6E),Fos,Npas4,and Egr1expression was markedly upregulated upon stimulation and returned to base-line levels by2hr post-stimulation in untreated controls.Incuba-tion with NU7026had 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 at2hr post-stimulation relative to untreated Cell161,1592–1605,June18,2015a2015Elsevier Inc.1599
A B C
D E
F
Figure5.Activity-Induced DNA DSBs Occur within Topological Domains De?ned by CTCF Binding
(A)Topo II b peaks in NMDA-treated samples were categorized into two groups—Class I represents new Topo II b peaks that appear after NMDA treatment and Class II denotes Topo II b peaks that are present under both basal and NMDA-treated conditions.Aggregate plots of g H2AX signals within a4kb window relative to Topo II b peaks in each class were then generated as in Figure4F.
(B)Motif scans at genome-wide Topo II b binding sites under basal conditions revealed a strong enrichment for the CTCF transcription factor binding site motif (CTCF_1;https://www.doczj.com/doc/e517757760.html,/$pouyak/motifs-table/)in the vicinity of Topo II b peaks.The plot denotes the disposition of input-normalized Topo II b signals relative to CTCF sites that displayed Topo II b peaks in their vicinity.Dashed line denotes the pro?le of Topo II b under basal conditions(control),whereas the solid line indicates Topo II b pro?les in NMDA-treated samples.
(C)Publicly available CTCF ChIP-seq datasets from the cortical plate of8week-old mice(GEO:GSM918727)were used to determine the overlap of CTCF and Topo II b binding pro?les at CTCF motifs that were enriched for Topo II b peaks in(B).The gray bar in the middle indicates the width of the CTCF peak at these sites. Dashed line denotes the pro?le of Topo II b under basal conditions(control),whereas the solid line indicates Topo II b pro?les in NMDA-treated samples.
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controls(Figure6F),although the expression of these genes at2hr was still lower than their peak induction levels at30min post-stim-ulation(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 II b in activity-induced DSB formation and early-response gene expression,we infected cultured pri-mary neurons with lentiviral vectors carrying shRNAs against Top2b.qRT-PCR experiments performed one week after the len-tiviral infections using two distinct shRNAs revealed that both shRNAs were able to knockdown Top2b expression by at least 50%(Figure S6B).To examine the effects of Top2b knockdown on DSB formation,we assessed g H2AX enrichment within the exons of early-response genes after NMDA treatment using ChIP-qPCR.Whereas NMDA treatment caused a robust increase in g H2AX levels in the exons of Fos,Npas4,and Egr1in neurons infected with a scrambled shRNA,this increase was attenuated in neurons infected with Top2b shRNAs(Figure6G).In addition to this,qRT-PCR experiments following NMDA treatment of cultured primary neurons revealed that whereas neurons infected with scrambled shRNA showed a signi?cant induction of Fos and Npas4transcripts following activity stimulation,the induction of these genes was severely attenuated in neurons infected with Top2b shRNAs(Figure6H).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 S6C).These results suggest that Topo II b 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 II b affects synaptic functions, we stereotactically injected lentiviral vectors carrying Top2b shRNAs into the CA1region of the mouse hippocampus.Four weeks after viral injections,we performed extracellular record-ings in acute hippocampal slices.As assessment of basal synap-tic transmission revealed no signi?cant differences between control slices infected with a scrambled shRNA and slices in-fected with Top2b shRNAs(Figure S6D).However,theta-burst stimulation(TBS)-induced LTP from Schaffer collateral-CA1syn-apses was severely impaired in slices infected with Top2b shRNAs(Figure6I).While the slices transduced with scrambled shRNA exhibited200%fEPSP over baseline,shRNA#1trans-duced slices showed130%and shRNA#2transduced slices showed150%fEPSP over baseline over the time course of 60min following theta-burst stimulation(Figure6I).These results are consistent with a role for Topo II b in the expression of early-response genes and maintaining synaptic function.
To further understand the connections between activity-induced DSBs,Topo II b,and early-response gene expression, we performed RNA-seq after NMDA treatment of cultured primary neurons(Table S5).Analysis of differentially expressed genes af-ter NMDA treatment revealed that only the early-response genes,Fos,Npas4,FosB,and Egr1,show elevated g H2AX signals. Furthermore,only early-response genes were upregulated by both etoposide and NMDA treatments(Figures1B and1D,and Tables S1and S5).These results suggest that activity-dependent DSB formation selectively promotes the expression of early-response genes.Finally,we tested whether DSB formation in the context of Topo II b knockdown could restore the expression of early-response genes.To this end,we infected cultured primary neurons with lentiviral vectors carrying shRNAs against Top2b, together with lentiviral vectors carrying either Cas9and sgRNAs against the Fos promoter(Cas9-Fos/CRISPR)or Cas9and sgRNAs against a different region(Cas9-control/Control).We then treated neurons with NMDA and compared Fos levels under these conditions.Consistent with our earlier observations(Fig-ure6C),neurons infected with both Cas9-Fos and scrambled shRNA were able to induce Fos under basal conditions(Figure6J). Likewise,neurons infected with Cas9-control and Top2b shRNA showed reduced Fos expression following NMDA treatment(Fig-ure6J).In contrast,neurons infected with a combination of Cas9-Fos and Top2b shRNA were able to successfully upregulate Fos expression following NMDA treatment(Figure6J),indicating that DSB formation can restore the expression of early-response genes in the absence of Topo II b.Taken together,our results sug-gest that Topo II b-mediated DSBs facilitate the expression of early-response genes in response to neuronal activity.
DISCUSSION
DSBs Facilitate the Expression of Early-Response Genes
The promoters of early-response genes already display the major hallmarks of transcriptionally active genes under basal conditions,including RNAPII bound at the TSS(Kim et al., 2010).Moreover,neuronal activity only minimally impacts the pro?les of histone methylation and transcription factor binding at these promoters.These observations raise important ques-tions about how the expression of early-response genes is cur-tailed under basal conditions and how neuronal stimulation causes these genes to be expressed so instantly.
Our results provide three lines of evidence that suggest that DSB formation could help override the impediments to early-response gene expression:(1)DSB formation in the promoters of early-response genes using either etoposide(Figures1D, 4F,and S4B)or CRISPR-Cas9(Figures6B and6C)is suf?cient to induce the expression of early-response genes even in the absence of neuronal activity,(2)inhibition of DSB repair results in persistent expression of early-response genes(Figure6F), and(3)Topo II b knockdown precludes the expression of early-response genes following neuronal activity;however,gene expression can be restored under these conditions by the gener-ation of targeted DSBs(Figure6J).
(D)A similar analysis to(C)was conducted using another publicly available CTCF ChIP-seq dataset from mouse neural progenitors(Phillips-Cremins et al.,2013). Grey bar and lines are as in(C).
(E)Aggregate plots of input-normalized CTCF(violet)(Phillips-Cremins et al.,2013)and g H2AX(orange)signals within a4kb window relative to the transcription start site(TSS)and transcription termination site(TTS)(gray box)of the loci that show increased g H2AX intensity following NMDA treatment.
(F)Aggregate plots of input-normalized CTCF(violet)(Phillips-Cremins et al.,2013)and g H2AX(blue)signals within a4kb window relative to the TSS and TTS (gray box)of the loci that show increased g H2AX intensity following etoposide treatment.
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Figure6.Topo II b-Mediated DNA DSBs Govern the Expression of Early-Response Genes Following Neuronal Activity
(A)Schematic of Fos and Npas4genes 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 Npas4upstream 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 measured16hr after transfection.As a control,luciferase reporter constructs in each case were transfected with Cas9and 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 Cas9and sgRNAs directed to either the Fos or the Npas4promoter.RNA was extracted 8days post-infection and the expression of Fos and Npas4was determined relative to neurons infected with sgRNAs directed to the Bdnf promoter using qRT-PCR(n=3,**p<0.01,two-tailed t test).
(D)Cultured primary neurons were treated with NMDA as before and allowed to recover in NMDA-free media.Topo II b ChIP was then performed at the indicated times and the ampli?cation of the Fos promoter was assessed as in Figure3A(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 Egr1was assessed using qRT-PCR(n=4,***p<0.001,one way-ANOVA).
(F)Cultured primary neurons were incubated with a speci?c inhibitor of DNA-PK(NU7026)for1hr,following which neurons were treated with NMDA and allowed to recover in NMDA-free media.RNA was extracted2hr after the initial NMDA treatment(time=0,x axis),and the levels of Fos,Npas4,and Egr1was assessed relative to neurons treated with NMDA in the absence of NU7026using 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#1or shRNA#2).One week after the infection,neurons were treated with NMDA(50m M)for10min followed by recovery in NMDA-free media for an additional10min.Enrichment of g H2AX within exons of Fos,Npas4,and Egr1was assessed using ChIP.As a control,the Fos promoter region was also probed (n=3,**p<0.01,two-way ANOVA).
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Topological Factors Constrain the Expression of
Early-Response Genes
The involvement of Topo II b suggests the existence of topolo-gical constraints that could govern the expression of early-response genes.However,the precise nature of these topolog-ical constraints is presently unclear.Topo II b activity upstream of the TSS is generally important for transcription elongation (Teves and Henikoff,2014).In fact,Topo II b is known to bind to the50ends of actively transcribed neurodevelopmental genes and promote their expression(Lyu et al.,2006;Tiwari et al., 2012).As mentioned above,the promoters of early-response genes already contain paused RNAPII under basal conditions, and activity-induced DSB formation could be a way to rapidly resolve topological barriers to RNAPII movement.Torsional stress generated during transcription initiation at these pro-moters could cause Topo II b recruitment under basal conditions. Notably however,the recruitment of Topo II b to the promoters of early-response genes under basal conditions does not result in the formation of DSBs,and the potential relaxation of supercoils by Topo II b under basal conditions by itself is insuf?cient to induce the expression of early-response genes.Furthermore, blocking transcription elongation does not affect activity-depen-dent DSB formation(Figure S5C).Despite these,the possibility that neuronal activity spawns new transcription-related torsional forces that induce DSB formation by Topo II b cannot be ruled out.
Separate from transcription-related torsional factors however, the TSS of genes that incur activity-dependent DSBs are charac-terized by their close proximity to CTCF binding sites compared to the TSS of other genes(Figure S5E),and Topo II b binding is preferentially enriched at CTCF binding sites(Figure5).We believe these observations provide insights into a distinct topo-logical constraint to early-response gene expression.CTCF-mediated chromatin loops create topological barriers that govern the interactions between distinct regulatory regions, such as promoters and enhancers(Ong and Corces,2014). Enhancer-promoter interactions are known to be essential for the expression of early-response genes.Enhancers of early-response genes are also pre-bound by SRF and CREB and re-cruit RNAPII and CBP following neuronal activity,and several studies indicate that RNAPII recruited to enhancer loci might be transferred to promoters following activity stimulation(Kim et al.,2010;Koch et al.,2008).Within this context,the formation of Topo II b-mediated DSBs at CTCF binding sites in an activity-dependent manner constitutes an attractive model that would rapidly dissolve topological constraints to enhancer-promoter interactions and instantly stimulate the expression of early-response genes.Implications of Activity-Dependent DSB Formation in Neurodegeneration
Because activity-dependent DSBs form in the regulatory regions of genes that govern crucial neuronal functions,the dynamics of their formation and repair is likely to have important physiological and pathological implications.Interestingly,androgen signaling was previously shown to induce Topo II b-mediated DSBs that results in oncogenic rearrangements observed in human prostate cancer(Haffner et al.,2010).These observations raise the intriguing question of whether unrepaired or erroneously re-paired DSBs produced in response to neuronal activity could contribute to the development of neurological disorders. Elevated DNA DSBs have been reported in a variety of congenital and age-related neurological disorders(Madabhushi et al.,2014; McKinnon,2013).However,the relevant sources that lead to DSB accumulation in these disorders remain unknown.Interest-ingly,it was recently reported that amyloid b accumulation, which is a hallmark of Alzheimer’s disease,can exacerbate the accumulation of DSBs that are produced during normal physio-logical neuronal activities in mice(Suberbielle et al.,2013).It would be interesting to test whether elevated DSBs in this mouse model,as well as in other mouse models and postmortem brains of patients with neurodegenerative disorders,overlap with the sites of activity-induced DNA DSBs.
The enrichment of TDP2at the promoters of early-response genes(Figure S3B)suggests that activity-induced DSBs are likely repaired error-free despite the utilization of the NHEJ pathway(Figure6F).In cells lacking TDP2,Topo II b-mediated DSBs are repaired using alternative mechanisms of end joining that are dependent on ATM(A′lvarez-Quilo′n et al.,2014).How-ever,such end-processing steps could be mutagenic.It was recently reported that individuals carrying mutations in TDP2 manifest with intellectual disability,epilepsy,and ataxia,and that the loss of TDP2in cultured human cells and postmitotic neurons caused hypersensitivity to Topo II b-mediated DSBs (Go′mez-Herreros et al.,2014).An assessment of TDP2activity in various neurodegenerative mouse models that are character-ized by elevated levels of DNA DSBs would likely provide further insights in this direction.
EXPERIMENTAL PROCEDURES
Neuronal Cultures and Treatments
Dissociated cortical neurons from E16Swiss-Webster mice were plated at a density of12.5million cells/plate in10cm plates.The plates were coated beforehand by incubation with poly-D-lysine(0.5mg/ml)and laminin (0.005mg/ml)for1hr at37 C,followed by washing twice with dH2O.Neurons were maintained in neurobasal media(GIBCO)and supplemented with L-glutamine,penicillin/streptomycin,and B27.
(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 Npas4were 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/6mice.Four weeks after the injections,acute hippocampal slices were prepared and LTP was induced by33TBS at the Schaffer collateral-CA1synapses.Sample traces represent fEPSPs1min before(gray) and1hr after(black)TBS.Scale bars,1mV and20ms(5–6slices per animal,3animals per group,*p<0.05,one-way ANOVA).
(J)Cultured primary neurons were infected with a combination of lentiviral vectors carrying Cas9and sgRNAs directed to the Fos promoter(CRISPR)together with Top2b shRNAs.Controls represent neurons infected with Cas9and sgRNAs directed to the Bdnf promoter together with scrambled shRNAs(control).One week after the lentiviral infections,neurons were treated with NMDA(50m M)for10min followed by recovery in NMDA-free media for10min.RNA was then extracted and Fos expression was assessed using qRT-PCR(n=3,*p<0.05one-way ANOVA).
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Neuronal activity was induced by treatment with indicated concentrations of potassium chloride(KCl),bicucullin(Bic),and N-methyl-D-aspartate (NMDA).For most experiments,cultured primary neurons were treated with50m M NMDA for10min followed by recovery in NMDA-free conditional media for an additional10min.Etoposide(Sigma)was at5m M,Olaparib (PARPi)(Selleck)at10m M,Ncs was at1m M,and NU7026(Calbiochem)was at10m M.
DNA Cleavage Assays
Topo II b-mediated DNA cleavage assays were essentially carried out as described(Nitiss et al.,2012).Brie?y,reaction mixtures(20m l)containing 10mM Tris(pH8),50mM NaCl,50mM KCl,5mM MgCl2,0.1mM EDTA,15m g/ml BSA,and10nM supercoiled plasmids(Fos-luc,Npas4-luc, D Fos-luc,and D Npas4-luc)were incubated with8units of recombinant Topo II b-His(LAE Biotech).Where indicated,etoposide was at0.2mM.Reac-tions were incubated at30 C for10min.DNA cleavage products were trapped by addition of2m l of10%SDS.Samples were then treated with1m l of250mM EDTA and2m l of1mg/ml proteinase K and incubated at37 C for45min. Samples were then loaded onto1%TAE agarose gels and electrophoresed at5V/cm for4hr.Gels were stained with1m g/ml ethidium bromide for 30min and visualized.
In Vivo Complex of Enzyme Assay
ICE assays were performed essentially as described(Nitiss et al.,2012). Cultured primary neurons were lysed with1%sarkosyl,and the DNA was sheared using a1ml syringe with a25G/8gauge needle.Lysates were passed 10times through the needle.Lysates(2ml)were then layered atop a2ml CsCl solution in13351mm polycarbonate centrifuge tubes(Beckman)and spun at 71,000rpm in an SLA-100.3rotor(Beckman)for12hr at room temperature. Pellets were washed with70%ethanol and resuspended in TE buffer(pH 7.5).Indicated amounts of DNA were applied to a nitrocellulose membrane us-ing a slot-blot apparatus(Bio-Rad)following manufacturer’s instructions.The membranes were then analyzed for Topo II b using standard western blotting procedures.
ChIP-Seq,RNA-Seq,ChIP-qPCR,Behavioral and
Elecrophysiological Paradigms
Please refer to Supplementary Information for details of these procedures.
A comprehensive list of primers used for these methods can be found in Table S6.
ACCESSION NUMBERS
The accession number for the ChIP-seq and RNA-seq data reported in this paper is GEO:GSE61887.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, six?gures,and six tables and can be found with this article online at http:// https://www.doczj.com/doc/e517757760.html,/10.1016/j.cell.2015.05.032.
ACKNOWLEDGMENTS
We would like to thank Dr.Yingxi Lin(Massachusetts Institute of Technology) for kindly providing the Fos and Npas4luciferase reporter constructs.This work was supported by the Lord Foundation Fellowship to R.T.S,the NIH/ NHGRI(R01HG004037-07and RC1HG005334)awards to M.K.,and NIH R01AG046174,the Glenn Foundation,and the Belfer Neurodegeneration Con-sortium grants to L.-H.T.
Received:September26,2014
Revised:January19,2015
Accepted:April7,2015
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年份平均值标准差变异系数 1966-1970-4.8213.35-2.77 下表给出了某气象台站五年的月平均气温， （1）试计算每一个年度的变异系数(注：结果是五个变异系数) （2）把1966—1970年各月的月平均气温数据，尾首相接后产生一个新 的时间序列，再计算变异系数（注：结果是一个变异系数） （3）如果把摄氏温度转化为华氏温度，再计算变异系数；那么结果与 用摄氏温度的数据计算的结果，相同吗？如果不同，究竟哪种答案是正 确的，产生的原因是什么？ 某气象台站五年的月平均气温（单位：摄氏度）年份一月二月三月四月五月六月七月八月 1966-21.6-21.7-13.1-3.1 3.09.710.011.5 1967-35.2-26.9-12.40.9 6.59.59.88.9 1968-24.0-24.6-5.50.0 6.38.310.49.3 1969-26.0-23.6-8.1 1.0 5.68.810.79.3 1970-28.2-21.9-10.10.9 5.18.28.29.6 （1）（3）根据变异系数公式计算每一年的变异系数如下： 年份变异系数(摄氏温 度)变异系数（华氏温度） 1966-2.76 1.02 1967-2.62 1.33 1968-2.77 1.08 1969-3.400.92 1970-2.90 1.07 （2）把1966—1970年各月的月平均气温数据，尾首相接后产生一个新的时间序列，再计算变异系数为： 分析结果：
通过查阅相关资料可知变异系数和极差、标准差和方差一样，都是反映数据离散程度的绝对值。其数据大小不仅受变量值离散程度的影响，而且还受变量值平均水平大小的影响。从上面的图表可以看出摄氏温度计算出来的变异系数都为负值，而通过华氏温度计算出来的变异系数都为正值，两者处理结果不同主要是将摄氏温度转换为华氏温度并不是一个比例变换。我认为两者方法都可取。
第三章 平均数、标准差与变异系数 本章重点介绍平均数（mean ）、标准差（standard deviation ）与变异系数（variation coefficient ）三个常用统计量，前者用于反映资料的集中性，即观测值以某一数值为中心而分布的性质；后两者用于反映资料的离散性，即观测值离中分散变异的性质。 第一节 平均数 平均数是统计学中最常用的统计量，用来表明资料中各观测值相对集中较多的中心位置。在畜牧业、水产业生产实践和科学研究中，平均数被广泛用来描述或比较各种技术措施的效果、畜禽某些数量性状的指标等等。平均数主要包括有算术平均数（arithmetic mean ）、中位数（median ）、众数（mode ）、几何平均数（geometric mean ）及调和平均数（harmonic mean ），现分别介绍如下。 一、算术平均数 算术平均数是指资料中各观测值的总和除以观测值个数所得的商，简称平均数或均数，记为x 。算术平均数可根据样本大小及分组情况而采用直接法或加权法计算。 （一）直接法 主要用于样本含量n ≤30以下、未经分组资料平均数的计算。 设某一资料包含n 个观测值：x 1、x 2、…、x n ，则样本平均数x 可通过下式计算： n x n x x x x n i i n ∑== +++=1 21Λ （3-1） 其中，Σ为总和符号； ∑=n i i x 1表示从第一个观测值x 1 累加到第n 个观测值x n 。当∑=n i i x 1 在意义上已明确时，可简写为Σx ，（3-1）式即可改写为： n x x ∑= 【例3.1】 某种公牛站测得10头成年公牛的体重分别为500、520、535、560、585、 600、480、510、505、490（kg ），求其平均体重。 由于Σx =500+520+535+560+585+600+480+510+505+490=5285，n =10 代入（3—1）式得： .5(kg)52810 5285∑=== n x x 即10头种公牛平均体重为528.5 kg 。 （二）加权法 对于样本含量n ≥30以上且已分组的资料，可以在次数分布表的基础上采用加权法计算平均数，计算公式为：
全区可采：全部或基本全部可采； 大部分可采：局部可采～全区可采； 局部可采：有1/3左右分布比较集中的面积。 零星可采：面积很小，或分布零星，不便或不能被开发利用。 厚度：全层厚度、纯煤厚度、采用厚度（即估算厚度）。 全层厚度：包括夹矸，但不包括岩浆岩。用于研究煤层沉积环境、赋存规律、煤层对比。 采用厚度：即估算厚度，用于煤层可采程度评价(全区可采、大部分可采、局部可采)和估算资源储量。
钻孔控制可采、局部可采煤层情况一览表表4-2-3
一、采用厚度与全层厚度的区别 采用厚度主要用于煤层可采程度评价和估算煤层的资源量。 在研究煤层的沉积环境、赋存规律、煤层对比时，以考虑煤层的全层厚度为宜。 二、含煤系数： 含煤系数= 各煤层平均煤厚之和 ×100% 地层总厚度 三、可采煤层的煤厚与平均煤厚： 可采煤层的煤厚与平均煤厚应包括夹矸在内，因为在研究煤层的沉积环境、赋存规律、煤层对比时，以考虑煤层的全层厚度为宜。沉缺点、冲刷点、火侵点煤厚为0，当有岩浆岩夹矸时，应将岩浆岩夹矸扣除在外。 三、可采煤层的可采性指数(Km 为小数，一般取小数点后两位)： 可采性指数（Km ）= 可采点数（n ′） 见煤点数（n ） n ——井田内参与煤厚评价的见煤点总数（不包括沉缺、冲刷、火侵，要求分布均匀，有代表性） n ′——煤层采用厚度≥最低可采厚度的见煤点数 注：沉缺点、冲刷点、火侵点为非见煤点，不参与统计 四、可采煤层的煤厚变异系数(r 为百分数，一般取不保留小数)： （注：这里用的煤厚是指的煤层全厚度） %100?=M S r M ——井田内的平均煤厚 S ——均方差 煤层平均厚度公式 n M M M M M n ++++= 321 1 ) (1 2 --= ∑=n M M S n i i
二、权重的确定方法 在统计理论和实践中，权重是表明各个评价指标（或者评价项目）重要性的权数，表示各个评价指标在总体中所起的不同作用。权重有不同的种类，各种类别的权重有着不同的数学特点和经济含义，一般有以下几种权重。 按照权重的表现形式的不同，可分为绝对数权重和相对数权重。相对数权重也称比重权数，能更加直观地反映权重在评价中的作用。 按照权重的形成方式划分，可分为人工权重和自然权重。自然权重是由于变换统计资料的表现形式和统计指标的合成方式而得到的权重，也称为客观权重。人工权重是根据研究目的和评价指标的内涵状况，主观地分析、判断来确定的反映各个指标重要程度的权数，也称为主观权重。 按照权重形成的数量特点的不同划分，可分为定性赋权和定量赋权。如果在统计综合评价时，采取定性赋权和定量赋权的方法相结合，获得的效果更好。 按照权重与待评价的各个指标之间相关程度划分，可分为独立权重和相关权重。 独立权重是指评价指标的权重与该指标数值的大小无关，在综合评价中较多地使用独立权重，以此权重建立的综合评价模型称为“定权综合”模型。 相关权重是指评价指标的权重与该指标的数值具有函数关系，例如，当某一评价的指标数值达到一定水平时，该指标的重要性相应的减弱；或者当某一评价指标的数值达到另一定水平时，该指标的重要性相应地增加。相关权重适用于评价指标的重要性随着指标取值的不同而发生变化的条件下，基于相关权重建立的综合评价模型被称为“变权模型”。比如评估环境质量多采用“变权综合”模型。 确定权重的方法较多，这里介绍统计平均法、变异系数法和层次分析法，这些也是实际工作种常用的方法。 (一) 统计平均法 统计平均数法（Statistical average method）是根据所选择的各位专家对各项评价指标所赋予的相对重要性系数分别求其算术平均值，计算出的平均数作为各项指标的权重。其基本步骤是： 第一步，确定专家。一般选择本行业或本领域中既有实际工作经验、又有扎实的理论基础、并公平公正道德高尚的专家； 第二步，专家初评。将待定权数的指标提交给各位专家，并请专家在不受外界干扰的前提下独立的给出各项指标的权数值； 第三步，回收专家意见。将各位专家的数据收回，并计算各项指标的权数均值和标准差；
变异系数 变异系数又称“标准差率”，是衡量资料中各观测值变异程度的另一个统计量。当进行两个或多个资料变异程度的比较时，如果度量单位与平均数相同，可以直接利用标准差来比较。如果单位和（或）平均数不同时，比较其变异程度就不能采用标准差，而需采用标准差与平均数的比值（相对值）来比较。 标准差与平均数的比值称为变异系数，记为C.V 。变异系数可以消除单位和（或）平均数不同对两个或多个资料变异程度比较的影响。 标准变异系数是一组数据的变异指标与其平均指标之比，它是一个相对变异指标。 变异系数有全距系数、平均差系数和标准差系数等。常用的是标准差系数，用C V(Coefficient of Variance)表示。 CV(Coefficient of Variance):标准差与均值的比率。 用公式表示为：CV ＝σ/μ 作用：反映单位均值上的离散程度，常用在两个总体均值不等的离散程度的比较上。若两个总体的均值相等，则比较标准差系数与比较标准差是等价的。 变异系数又称离散系数。 cpa 中也叫“变形系数” 1．标准差是用来反映各个数据值与数据均值的偏离程度的。标准差可以用来评价同一指标的各数据与这一指标数据平均值的偏离程度，即数据是否集中。标准差的值越大，就说明各个数据偏离均值的程度越大，那么均值对所有数据的代表程度越小。反之，标准差的值越小，就说明各个数据偏离均值的程度越小，那么均值对所有数据的代表程度越大。 标准差的计算： 假设标准差为S 。 对于未分组的原始数据，其标准差的计算公式为： n ) X X (S 2 n 1i i ∑-=＝（n>=30） 1n ) X X (S 2i -∑-=（n<30）
excel变异系数函数的计算方法 在Excel中经常会利用到函数进行数据的统计计算，虽然变异很少求到，但也会用到，下面是小编带来的关于excel变异系数函数的计算方法的内容，欢迎阅读! excel变异系数函数的计算方法 变异系数(Coefficient of Variation)又称标准差率，是衡量资料中各观测值变异程度的另一个统计量。当进行两个或多个资料变异程度的比较时，如果度量单位与平均数相同，可以直接利用标准差来比较。如果单位和(或)平均数不同时，比较其变异程度就不能采用标准差，而需采用标准差与平均数的比值(相对值)来比较。 标准差与平均数的比值称为变异系数，记为C.V。变异系数可以消除单位和(或)平均数不同对两个或多个资料变异程度比较的影响. 变异系数越小，变异(偏离)程度越小，风险也就越小;反之，变异系数越大，变异(偏离)程度越大，风险也就越大。 变异系数的计算公式为：变异系数CV =标准偏差/ 平均值 如表：为某公司的用户使用的连续3个月的使用流量状况，如何求各用户的变异系数? 用户T-3月流量T-2月流量T-1月流量A0001283.3320.5273.3A0002102.1140.4180.3A000320.512.33 4.6A0004800.81029.8980.3A0005502.8321.3325.5A0006245.8
278.9296.4 A：主要解法如下： excel变异系数函数的计算方法1：首先使用AVERAGE函数求均值： =AVERAGE(B2:D2) excel变异系数函数的计算方法2：然后使用STDEV函数求标准差： =STDEV(B2:D2) excel变异系数函数的计算方法3：最后得到变异系数:CV=标准差/均值 最终结果如图所示： 用户T-3月流量T-2月流量T-1月流量均值标准差变异系数A0001283.3320.5273.3292.3724.870.085A0002102.1140.4180. 3140.9339.100.277A000320.512.334.622.4711.280.502A00048 00.81029.8980.3936.97120.490.129A0005502.8321.3325.5383. 20103.600.270A0006245.8278.9296.4273.7025.700.094看了excel变异系数函数的计算方法还看了：1.浅谈小麦产量构成因素的相关性分析 2.阿莫西林的研究进展 3.论中国地区工业发展态势及政策导向
变异系数的意义 变异系数（又称离散系数）是概率分布离散程度的一个归一化量度。 变异系数只在平均值不为零时有定义，而且一般适用于平均值大于零的情况。变异系数也被称为标准离差率或单位风险。 变异系数只对由比率标量计算出来的数值有意义。举例来说，对于一个气温的分布，使用开尔文或摄氏度来计算的话并不会改变标准差的值，但是温度的平均值会改变，因此使用不同的温标的话得出的变异系数是不同的。也就是说，使用区间标量得到的变异系数是没有意义的。 在概率论和统计学中，变异系数，又称“离散系数”（英文：coefficient of variation），是概率分布离散程度的一个归一化量度，其定义为标准差与平均值之比： 变异系数（coefficient of variation）只在平均值不为零时有定义，而且一般适用于平均值大于零的情况。变异系数也被称为标准离差率或单位风险。 变异系数只对由比率标量计算出来的数值有意义。举例来说，对于一个气温的分布，使用开尔文或摄氏度来计算的话并不会改变标准差的值，但是温度的平均值会改变，因此使用不同的温标的话得出的变异
系数是不同的。也就是说，使用区间标量得到的变异系数是没有意义的。 2基本含义 变异系数 一般来说，变量值平均水平高，其离散程度的测度值越大，反之越小。 变异系数是衡量资料中各观测值变异程度的另一个统计量。当进行两个或多个资料变异程度的比较时，如果度量单位与平均数相同，可以直接利用标准差来比较。如果单位和（或）平均数不同时，比较其变异程度就不能采用标准差，而需采用标准差与平均数的比值（相对值）来比较。标准差与平均数的比值称为变异系数，记为C·V。变异系数可以消除单位和（或）平均数不同对两个或多个资料变异程度比较的影响。 变异系数的计算公式为：变异系数C·V =（标准偏差SD / 平均值Mean ）× 100% 在进行数据统计分析时，如果变异系数大于15%，则要考虑该数据可能不正常，应该剔除。 3举例