Gene activation of SMN by selective disruption oflncRNA-mediated recruitment of PRC2 for thetreatment of spinal muscular atrophyCaroline J. Wooa,1, Verena K. Maiera, Roshni Daveya, James Brennana, Guangde Lia, John Brothers IIa, Brian Schwartza,Susana Gordoa, Anne Kaspera, Trevor R. Okamotob, Hans E. Johanssonb, Berhan Mandefroc,d, Dhruv Sareenc,d,e,Peter Bialeka, B. Nelson Chaua, Balkrishen Bhata, David Bullougha, and James Barsouma

aRaNA Therapeutics, Cambridge, MA 02139; bLGC Biosearch Technologies, Petaluma, CA 94954; cBoard of Governors–Regenerative Medicine Institute,Cedars–Sinai Medical Center, Los Angeles, CA 90048; dInduced Pluripotent Stem Cell Core, The David and Janet Polak Stem Cell Laboratory, Cedars–SinaiMedical Center, Los Angeles, CA 90048; and eDepartment of Biomedical Sciences, Cedars–Sinai Medical Center, Los Angeles, CA 90048

Edited by Robert E. Kingston, Massachusetts General Hospital/Harvard Medical School, Boston, MA, and approved January 10, 2017 (received for reviewOctober 4, 2016)

Spinal muscular atrophy (SMA) is a neurodegenerative diseasecharacterized by progressive motor neuron loss and caused bymutations in SMN1 (Survival Motor Neuron 1). The disease severityinversely correlates with the copy number of SMN2, a duplicatedgene that is nearly identical to SMN1. We have delineated a mech-anism of transcriptional regulation in the SMN2 locus. A previouslyuncharacterized long noncoding RNA (lncRNA), SMN-antisense 1(SMN-AS1), represses SMN2 expression by recruiting the PolycombRepressive Complex 2 (PRC2) to its locus. Chemically modified oligo-nucleotides that disrupt the interaction between SMN-AS1 and PRC2inhibit the recruitment of PRC2 and increase SMN2 expression in pri-mary neuronal cultures. Our approach comprises a gene-up-regulationtechnology that leverages interactions between lncRNA and PRC2.Our data provide proof-of-concept that this technology can be usedto treat disease caused by epigenetic silencing of specific loci.

spinal muscular atrophy | lncRNA | PRC2 | SMN

Spinal muscular atrophy is the leading genetic cause of infantmortality and is caused by deletions or mutation of Survival

Motor Neuron 1 (SMN1) (1). Unique to humans, SMN1 is dupli-cated in the genome as SMN2, which is nearly identical in se-quence. However, a C-to-T point mutation in exon 7 of SMN2results in preferential skipping of this exon during pre-mRNAsplicing and production of a truncated and unstable protein. Asmall fraction (10–20%) of pre-mRNA transcribed from SMN2is spliced correctly to include exon 7 and produces a full-lengthSMN (SMN-FL, inclusive of exon 7) that is identical to theSMN1 gene product (2–4).Spinal motor neurons are highly sensitive to SMN1 deficiency,

and their premature death causes motor function deficit in SMApatients (5, 6). The SMN2-derived SMN protein can extend spinalmotor neuron survival, yet insufficient levels of SMN eventuallylead to cell death. Overall, SMA patients with higher SMN2 ge-nomic copy number have a less severe disease phenotype (7, 8).Type 0 or I patients, carrying one or two copies of SMN2, showonset of SMA within a few months of life with a life expectancy ofless than 2. In contrast, type III and IV patients, carrying three ormore copies, respectively, show juvenile or adult onset and slowerdisease progression (9). As further genetic evidence, SMA mousemodels have been produced in which smn1−/− mice, which wouldotherwise be embryonic lethal (10), can be rescued in the presenceof high copy numbers of the human SMN transgene (11–13).Similar to the human disease spectrum, increased copy number ofa human SMN transgene is inversely associated with decreaseddisease severity and mortality. We reasoned that increasing SMN2transcription could phenocopy the beneficiary effect of SMN2gene amplification and compensate for SMN1 deficiency. In ad-dition, SMN1 heterozygotes are asymptomatic, whereas affectedhomozygotes have 10–20% of normal SMN levels. Therefore, we

predict that modest SMN2 up-regulation will provide significanttherapeutic benefit. Here, we establish that PRC2 interacts with along noncoding RNA (lncRNA) transcribed within the SMN2locus and regulates SMN2 expression through PRC2-associatedepigenetic modulation. Furthermore, we demonstrate that we canselectively up-regulate SMN2 expression by interrupting thelncRNA-mediated recruitment of PRC2 to the SMN2 locus. Suchan approach represents a therapeutic strategy for SMA and po-tentially can be used to elevate the expression of target genes invarious human disease settings.

ResultsPRC2 Modulates SMN2 Expression. Analysis of publicly availablechromatin immunoprecipitation (ChIP) sequencing data from theENCODE consortium and the Broad Institute (genome.ucsc.edu/)(14, 15) suggests that PRC2 is associated with SMN2 in differentcell types to varying degrees, most notably with the HepG2 cells(Fig. 1A). In addition, ChIP sequencing data from the NIHRoadmap Epigenome Consortium (16) suggests that H3K27me3,the hallmark of PRC2 activity, is associated with SMN2 in humanfetal brain (SI Appendix, Fig. S1). To determine whether disruption

Significance

Autosomal recessive mutations or deletions of the gene SurvivalMotor Neuron 1 (SMN1) cause spinal muscular atrophy, a neuro-degenerative disorder. Transcriptional up-regulation of a nearlyidentical gene, SMN2, can functionally compensate for the loss ofSMN1, resulting in increased SMN protein to ameliorate the dis-ease severity. Here we demonstrate that the repressed state ofSMN2 is reversible by interrupting the recruitment of a repressiveepigenetic complex in disease-relevant cell types. Using chemicallymodified oligonucleotides to bind at a site of interaction on a longnoncoding RNA that recruits the repressive complex, SMN2 isepigenetically altered to create a transcriptionally permissive state.

Author contributions: C.J.W., B.S., S.G., H.E.J., D.S., P.B., B.N.C., D.B., and J. Barsoum de-signed research; C.J.W., V.K.M., R.D., J. Brennan, G.L., B.S., S.G., A.K., T.R.O., H.E.J., B.M.,and P.B. performed research; D.S., B.N.C., and B.B. contributed new reagents/analytictools; C.J.W., V.K.M., R.D., J. Brennan, J. Brothers, B.S., S.G., A.K., T.R.O., H.E.J., P.B., andB.N.C. analyzed data; and C.J.W. wrote the paper.

Conflict of interest statement: C.J.W., V.K.M., R.D., J. Brennan, G.L., J. Brothers, B.S., S.G.,A.K., P.B., B.N.C., B.B., D.B., and J. Barsoum declare a financial interest in the body of workgenerated as shareholders and employees of RaNA Therapeutics.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE83549).1To whom correspondence should be addressed. Email: cwoo@ranarx.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616521114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616521114 PNAS | Published online February 13, 2017 | E1509–E1518

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of PRC2 activity could lead to increases in SMN2 expression, EZH1and EZH2 mRNAs were knocked down in the SMA fibroblast cellline, GM09677, using antisense oligonucleotides (ASOs) designedfor RNaseH-mediated degradation. Two days after transfection ofboth EZH1 and EZH2 gapmers, their respective mRNA levels weresignificantly decreased by ∼80% each and were associated with anincrease in SMN-FL mRNA as shown by reverse transcriptionquantitative PCR (RT-qPCR) (Fig.1B). We further analyzed theSMN1 and SMN2 loci (from here on collectively termed “SMNlocus”) for chromatin changes upon EZH1/EZH2 knockdown byperforming ChIP. Because SMN1 and SMN2 have >99% sequenceidentity (27,890 of 27,924 base pair match), it is not possible todistinguish between the two chromosomal locations here. We ob-served decreased association of EZH2 as well as decreasedH3K27me3 levels at the locus, without any changes in total H3 (Fig.1C). This suggests that PRC2 directly regulates the expression ofSMN in fibroblasts and potentially other cell types.

Identification of SMN-Antisense 1 at the SMN Locus. Detailed anal-ysis of RNA immunoprecipitation-sequencing (RIP-seq) datasetsrevealed a previously undescribed PRC2-interacting antisenseRNA within the mouse Smn locus (17). Here, we investigatedwhether the antisense transcript exists in humans and may have arole in PRC2-mediated SMN repression. Next-generation RNAsequencing revealed a lncRNA, which we call SMN-Antisense 1(SMN-AS1), that is transcribed from the SMN loci (Fig. 2A). Giventhe high sequence identity between the SMN1 and SMN2 loci, wepredict the lncRNA to be transcribed from both loci. As expected,SMN-AS1 was observed in both SMN1- and SMN2-mutated cell lines(Fig. 2B). Furthermore, SMN2 copy number was determined byqPCR for carrier and diseased cell lines (18), and we independentlydetermined the relative expression of SMN-AS1 and observed a directcorrelation (Fig. 2B). Northern blot analysis of human fetal brain andadult lung tissues revealed that SMN-AS1 is up to 10 kb long, isheterogeneous in size, and has differential expression between the

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Fig. 1. SMN2 locus is a target of PRC2 regulation. (A) UCSC Genome Browser screen shot of ChIP-sequencing mapped reads for EZH2, H3K27me3, and input at theSMN2 locus from HepG2 cells (reference genome GRCh37/hg19). The plot is a density graph of signal enrichment with a 25-bp overlap at any given site. (B) RT-qPCRfor EZH1 and EZH2 mRNA in SMA fibroblast line GM09677 after EZH1 and EZH2 knockdown by transfection of their respective targeting gapmer ASO for 2 d. RT-qPCR for EZH1, EZH2, and SMN-FLmRNA after EZH1 and EZH2 knockdown (mean ± SD; n = 3). *P < 0.05 with one-way ANOVA. (C) Schematic diagram of the SMN2locus with exons numbered above and with ChIP-qPCR primer positions below (red bars). ChIP-qPCR data for enrichment of EZH2, H3K27me3, and total H3 fromEZH1/EZH2 knockdown (green) compared with the lipid transfection control (purple) in the SMA fibroblasts (mean ± ; n = 3).

E1510 | www.pnas.org/cgi/doi/10.1073/pnas.1616521114 Woo et al.

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Fig. 2. Identification of a lncRNA at the SMN locus, SMN-AS1. (A) Mapping of SMN-AS1 positioned relative to the SMN genes. The red asterisk marks the locationof the C-to-T transition found in SMN2. AS3 and AS4 are Northern blot probes. (B) Correlation of expression and SMN copy number. SMN-AS1 relative expressionas measured by RT-qPCR (indicated on the left y axis, blue bars) and SMN2 genomic copy number as measured by digital qPCR (indicated on the right y axis, redbars) in SMA disease fibroblast lines and a carrier line by Zhong et al., 2011 (18). GM20384 cells lacking SMN2, but retaining SMN1, also express SMN-AS1.(C) Northern blot of human SMN-AS1 from human fetal brain and adult lung tissue detected with AS3 and AS4 probes. In the second Northern blot, WT and 5025SMA mouse brains were probed with AS3, showing the signal for SMN-AS1 is detected in the 5025 mouse harboring two copies of the human SMN locus. (D) RT-qPCR of SMN-AS1 and SMN-FLmRNA from 20 human tissue types with the fold change normalized to the expression level in the adrenal gland. (E) Strand-specificsingle-molecule RNA-FISH. Maximum intensity merge of widefield z stack in GM09677 SMA fibroblasts of the nascent SMN pre-mRNA (red, detected by a set ofintronic probes), the mature SMN mRNA (yellow), and the SMN-AS1 lncRNA (blue). Pre-mRNA signals are offset (up + left) and mature mRNA signals are offset(down + right) by 2 pixels to enable visualization. (F) Anti-SUZ12 nRIP of SMN-AS1with two primer sets (set 1 and set 2), TUG1 RNA, ANRIL RNA, 18S rRNA, GAPDHmRNA, beta-2-microglobulin (B2M) mRNA, and RPL19mRNA from SMA fibroblasts with enrichment shown as % input (mean ± SD; n = 3). IgG nRIP (blue) servedas the negative control for the SUZ12 nRIP (red). (G) RNA-EMSA of human PRC2 (EZH2/SUZ12/EED) combined with RepA I–IV, maltose-binding protein (MBP)(1–441), SMN-AS1 (PRC2 binding region), or SMN-AS1 (nonbinding region). Binding curves are displayed at Right (mean ± SD; n = 3).

Woo et al. PNAS | Published online February 13, 2017 | E1511

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two tissue types (Fig. 2C). To confirm the specificity of the SMN-ASprobe, we turned to a humanized SMA mouse model carrying twocopies of the human SMN2 genomic locus (5025 strain) (19). Com-paring the brain tissues from wild-type and 5025 mice, we observed asimilar set of transcripts in the SMN2-harboring transgenic miceas detected in the human fetal brain (Fig. 2C). By RT-qPCR, wedetected SMN-AS1 in patient cell lines, and the level of expres-sion correlated with SMN2 copy number (as determined by ref. 18)(Fig. 2B). In addition, we found that SMN2 mRNA and SMN-AS1expression is highly correlated, with highest levels in CNS tissues(Fig. 2D). Finally, strand-specific single-molecule RNA-fluorescent insitu hybridization (RNA-FISH) detected expression of SMN-AS1colocalized with the pre-mRNA transcript at the SMN locus (Fig. 2E)in all SMA fibroblasts (52 out of 52) that were imaged in three in-dependent experiments, suggesting that SMN-AS1 may function incis. We observed primarily one colocalized foci in the SMA fibro-blasts and a wild-type fibroblast line. Although the SMN1 and SMN2genes are separated linearly by 900 kb, their physical distance is un-known in vivo. Our data suggest that they are close enough inproximity in fibroblasts, such that single molecule RNA-FISH wouldnot be able to discern SMN1 from SMN2-derived signals. Together,these data demonstrate the presence of an antisense transcript in theSMN1 and SMN2 loci.

SMN-AS1 Binds PRC2. To investigate the role of SMN-AS1 in thePRC2-mediated epigenetic regulation of the SMN2 locus, weperformed native RIP (nRIP) using an antibody against the PRC2subunit SUZ12, followed by RT-qPCR with two distinct probe setsdirected to different regions of SMN-AS1. RIP-qPCR showed thatSMN-AS1 is strongly associated with PRC2 in SMA fibroblasts(Fig. 2F). The association was stronger than, or comparable to,that of well-established PRC2-interacting lncRNAs includingTUG1 (20) and ANRIL (21). Additionally, PRC2 did not associatewith highly expressed negative control transcripts such as GAPDHand RPL19. Similar results were observed with the nRIP for EZH2(SI Appendix, Fig. S1), further supporting the association of SMN-AS1 with PRC2. Because nRIP identifies both direct and indirectinteractions, we next performed RNA electrophoretic mobilityshift assays (RNA EMSAs), which specifically detect direct inter-actions. Using a 441-nt RNA containing the PRC2-interactingregion of SMN-AS1 (SMN-AS1, PRC2-binding region), as identi-fied by RIP-seq. (17), we observed that purified recombinant hu-man PRC2 (EED/SUZ12/EZH2) specifically changed its migration(Fig. 2G). Binding was concentration-dependent and was as robustas that of the 434-nt RepA RNA, a conserved domain of XISTRNA that is a well-documented PRC2-interacting lncRNA (17, 22,23). Dissociation constants (Kd) of both transcripts were estimatedto be 350–360 nM, suggesting that the association of this lncRNAwith PRC2 at this site is comparable to the RepA domain of XIST.As specificity controls, we observed a low level of backgroundbinding to a non–PRC2-interacting 441-nt region of the SMN-AS1transcript (SMN-AS1, nonbinding region) and to another non-specific mRNA of similar length, maltose-binding protein fromEscherichia coli (22). These data lead us to conclude that SMN-AS1lncRNA interacts directly and specifically with PRC2.

Blocking PRC2:SMN-AS1 Interaction Up-Regulates SMN2 and ProducesEpigenetic Changes. To investigate the effect of disruptingPRC2:SMN-AS1 interactions, we designed chemically modi-fied ASOs targeting the PRC2-binding region of the lncRNAfor hybridization via Watson–Crick complementarity pairing.Depending on the arrangement of DNA and locked nucleic acid(LNA)-modified nucleotides, such base pairing can lead to eitherRNaseH-mediated degradation of target RNAs or hindering ofthe interaction between target RNAs and their binding partners.For RNaseH-mediated degradation, a “gapmer”-formatted ASOcomposed of a central DNA segment greater than 6 nucleotides(i.e., gap) flanked by 2–4 LNA-modified nucleotides is required.

Such gapmer ASOs were used for knockdown of EZH1 and EZH2in earlier experiments (Fig. 1C). In contrast to the gapmer ar-rangement, a “mixmer”-formatted oligo lacks the central DNAcomponent by the introduction of interspersed chemically modi-fied nucleotides. It does not support the RNaseH-mediated deg-radation but rather functions as a steric blocker (24). We generatedmixmer oligos consisting of LNA interspersed with 2’-O-methylnucleotides for high-affinity binding to SMN-AS1. Oligos weredesigned to target regions enriched from EZH2-associated RNAsby RIP followed by next-generation RNA sequencing (17).Screening of mixmers led us to focus on one efficacious mixmer,

RN-0005 (Fig. 3A). Transfecting RN-0005 into SMA fibroblastssignificantly increased SMN-FL expression, whereas transfectingthe control oligo, RN-0012, which hybridizes to a region outside ofa PRC2 interaction domain, did not change SMN-FL expression(Fig. 3B). Consistently, nRIP showed that RN-0005, but not thecontrol oligo, disrupted the binding of PRC2 to SMN-AS1, asshown by RIP-qPCR (Fig. 3C). Furthermore, oligos targetingSMN-AS1 did not affect the interactions between PRC2 andANRIL, GAPDH, or RPL19 control RNAs. These results werealso observed when nRIP was performed using an antibody againstEZH2 (SI Appendix, Fig. S2). As expected, single-molecule RNA-FISH for the localization of SMN-AS1 after transfection with RN-0005 showed no change in both the abundance and the localizationof SMN-AS1 in ∼90% of cells examined (39 of 42 nuclei) per-formed in three independent experiments (SI Appendix, Fig. S3).Together, these results demonstrate that selective inhibition ofPRC2:SMN-AS1 interaction by a mixmer oligo leads to increasedSMN2 expression.To gain molecular insight into how the active oligo induced

SMN expression, we characterized the chromatin changes at theSMN locus in response to the disruption of PRC2:SMN-AS1interaction using ChIP. When SMA fibroblasts were treated withRN-0005, we observed a loss of EZH2 association as well asdecreased H3K27me3 along the SMN gene body (Fig. 3 D and E),suggesting that the oligo blocked the recruitment and activity ofPRC2. Furthermore, there was an increase in association of RNAPol II-phosphoSer2 (RNA polymerase II, phosphorylated at serine2) and elevated levels of H3K36me3, both of which indicate greatertranscriptional elongation (Fig. 3 F and G). By contrast, pan-H3levels were unaffected by treatment (Fig. 3H). H3K4me3, a markof transcription initiation, was enriched at the promoter in the lipidcontrol samples. Interestingly, H3K4me3 levels did not change atthe promoter with RN-0005 addition (Fig. 3I), suggesting that theincreased SMN mRNA levels may be occurring in a setting wherebasal levels of transcription exist. No changes in PRC2 associationwere observed at another well-established Polycomb target locus,HOXC13, upon treatment (Fig. 3J). We also performed ChIP onSMA fibroblasts that were treated with a splice-correcting oligo(SCO), which targets the pre-mRNA for exon 7 inclusion but doesnot alter the transcription rate at the SMN locus (SI Appendix, Fig.S4). No chromatin changes were observed. These data suggest thatPRC2 recruitment and histone methyltransferase activity at theSMN locus can be selectively inhibited by an oligo by mechanismsthat may include steric blocking of the specific PRC2:SMN-AS1interaction or disruption of a secondary structure within SMN-AS1that would be recognized by PRC2.

Blocking PRC2 Recruitment Results in SMN2 Up-Regulation inFibroblasts. We further characterized SMN2 mRNA up-regulation,which resulted from the disruption of the PRC2:SMN-AS1 in-teraction and the subsequent epigenetic changes at the SMNlocus. We used the GM09677 fibroblasts, which carry two copiesof the SMN2 gene and are homozygous for SMN1 exons 7 and 8deletion. RT-qPCR analyses with three different primer sets de-tected a concentration-dependent increase of various SMNmRNA transcripts, including all SMN isoforms (exon 1–2) as wellas isoforms including or excluding exon 7, SMN-FL, and SMNΔ7,

E1512 | www.pnas.org/cgi/doi/10.1073/pnas.1616521114 Woo et al.

respectively (Fig. 4A). Furthermore, overall SMN protein levelsalso increased, as shown by ELISA after 5 d of treatment, whichsupports the epigenetic evidence of increased transcription (Fig.4B). Western blot results revealed that this increase could be at-tributed to the 38-kDa SMN protein (Fig. 4C). Both quantificationof ELISA and Western analyses indicated an approximatelyfourfold protein up-regulation following treatment in SMA fi-broblasts with RN-0005. Taken together, blocking the interaction

of PRC2 with its recruiting lncRNA resulted in up-regulation ofboth SMN mRNA and protein.To determine how targeting the disruption of PRC2:SMN-AS1

interactions might affect PRC2 targets globally, we performedRNA sequencing from samples with specific disruption ofPRC2:SMN-AS1 (using RN-0005) or global inactivation of thePRC2 complex (using a SUZ12 gapmer). Treatment with eitherRN-0005 or the SUZ12 gapmer resulted in significant increases

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Fig. 3. PRC2 is associated with SMN-AS1 and selective dissociation leads to PRC2 loss and chromatin changes at the SMN locus. (A) Schematic diagram of theSMN2 locus with exons numbered above and mixmer oligo positions (squares) below. (B) RT-qPCR of SMN-FL mRNA after transfection with RN-0005 orRN-0012 in SMA fibroblasts for 3 d. (C) Anti-SUZ12 nRIP of SMN-AS1, ANRIL, GAPDH mRNA, and 18S rRNA from SMA fibroblasts after lipid (red) or RN-0005(green) or RN-0012 (blue) transfection; IgG RIP (purple) (mean ± SD; n = 3). *P < 0.05 using two-tailed Student’s t test. (D–I) ChIP at the SMN2 locus in GM09677SMA fibroblasts that were transfected with lipid (purple) or RN-0005 (green) using antibodies against (D) EZH2, (E) H3K27me3, (F) RNA polymerase IIphospho-Serine2, (G) H3K36me3, (H) pan-H3, and (I) H3K4me3 (mean ± SD; n = 3). (J) ChIP at the promoter of HOXC13, a PRC2-regulated gene, for EZH2,H3K27me3, RNA polymerase II, phospho-Serine 2 (RNA PolIIpS2), H3K36me3, H3, and H3K4me3, after transfection with lipid or RN-0005 in SMA fibroblasts.(mean ± SD; n = 3).

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in SMN mRNA levels compared with transfection control samples(Fig. 4D). Globally, there were approximately fourfold more geneexpression changes with the SUZ12 gapmer treatment than withRN-0005 treatment that had at least a 1.5-fold change (q < 0.05).This is depicted by a scatterplot of the moderated t statistics of thegene expression changes where for most genes, their individual

SUZ12 knockdown (KD) t statistic is usually larger than theirrespective RN-0005 t statistic. As this is a scatterplot encom-passing all genes and we expect that most genes do not changesignificantly or have a large magnitude of change, the two geneexpression profiles correlate overall. Here, a strong linear cor-relation was observed while simultaneously many more significant

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Fig. 4. Up-regulation of SMN expression upon RN-0005 treatment. (A) RT-qPCR of SMN (exon 1–2), SMN Δ7, and SMN-FL mRNA in GM09677 SMA fibroblasts(mean ± SD; n = 5). (B) Changes in total SMN protein levels after SMA fibroblasts were transfected with RN-0005 for 5 d (mean ± SD; n = 3), measured byELISA. (C) Western blot results for SMN and α-tubulin in SMA fibroblasts after SMA fibroblasts were transfected with RN-0005 for 5 d (mean ± SD; n = 2).(D) RT-qPCR of SMN-FL mRNA in GM09677 fibroblasts that were transfected with 15 nM RN-0005 or 15 nM SUZ12 gapmer ASO (mean ± SD; n = 4). *P < 0.05,**P < 0.01 using one-way ANOVA. A hexagonally binned scatterplot of the moderated t statistics of the 11,887 annotated genes tested for differentialexpression posttreatment with RN-0005 or the SUZ12 knockdown ASO. Each bin is colored by the number of genes that fall within it, showing the trend of RN-0005–treated t statistics (and those less significantly differentially expressed genes) generally being reduced compared with their SUZ12 knockdown ASOcounterpart t statistics. The Venn diagram shows the significant results (q < 0.10) of the pathway analysis using competitive gene set tests on 1,281 canonicalpathways after treatment with each oligo. Overlap required that a pathway was significantly affected in the same direction by both oligos. There is significantoverlap between the oligo treatments when tested with a hypergeometric test (P = 1.36e−11), however ∼4.5 times more pathway gene sets were significantlychanging with SUZ12 KD treatment.

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changes occurred with the SUZ12 gapmer treatment. Lookingcloser at expression profiles of genes neighboring SMN1 andSMN2, the nearest neighboring genes that changed significantlyin response to RN-0005 treatment were ADAMTS6 (upstream)and BDP1(downstream) at 4.6 Mb and 1.4 Mb away from SMN2,respectively. As the closest changed genes are greater than amegabase away, these data suggest PRC2:SMN-AS1 regulation islocalized to the SMN locus. In contrast, the nearest significantneighbor genes that changed after SUZ12 knockdown wereTAF9, 0.8 Mb upstream, and BDP1, 1.4 Mb downstream, ofSMN2. Furthermore, because both RN-0005 and the SUZ12 KDgapmer do affect SMN expression, we would expect to see po-tential overlap in downstream changes, which we confirm byidentifying 21 pathways overlapping between oligo treatments(only approximately 4–5 overlaps would be expected by chance ifthere was no relationship between the two treatments). However,we see far fewer pathways modulated significantly with RN-0005

treatment, suggesting fewer changes overall. Taken together, thissuggests that RN-0005 has a more localized effect than the moreglobal effects of knocking down PRC2. Pathway gene set analysesidentified significant pathways (10% false discovery rate) with eacholigo treatment, and although there were overlaps between thedatasets, many more pathways changed separately with SUZ12knockdown (Fig. 4D and SI Appendix, Fig. S5). This suggests thatalthough there is a relationship between the downstream geneseffected by modulating SMN expression, there are less overalldownstream changes when you up-regulate SMN expression withRN-0005 than when you target SMN through a global knockdownof PRC2.

Blocking PRC2 Recruitment Results in SMN2 Up-Regulation in NeuronalCultures. Although SMN expression is ubiquitous, its expression ishighest in the central nervous system (Fig. 2D) (25), particularly inspinal motor neurons where the disease is manifested (5, 6, 26).

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Fig. 5. Distinct mechanisms of SMN-FL mRNA generation can be complementary in neuronal cultures. (A, Left) RT-qPCR of SMN-FL mRNA in human SMA iPS-derived motor neuron cultures after gymnotic treatment with RN-0005, an inactive oligo, or an unrelated oligo at 5 and 20 μM for 11 d as a fold change fromuntreated cells (mean ± SD; n = 3). **P < 0.01 using one-way ANOVA. (Middle) RT-qPCR of SMN-FLmRNA in human SMA iPS-derived motor neuron cultures atday 7 after treatment with an EZH2 gapmer ASO (mean ± SD; n = 2). *P < 0.05 using one-way ANOVA. (Right) RT-qPCR for SMN-FL mRNA in human SMA iPS-derived motor neuron cultures after gymnotic treatment with RN-0005 at 20 μM for 3, 7, 9, or 11 d as a fold change from untreated cells (mean ± SD; n = 2).(B) Images of 5025 mouse cortical neurons at day 14 either mock-treated or treated with RN-0027 at 10 μM. (Scale bar, 100 μm.) (C) RT-qPCR of human SMN-FLmRNA relative to mouse gusb mRNA from the 5025 mouse cortical neurons that were treated with either 1.1, 3.3, or 10 μM RN-0027 (mean ± SD; n = 5) orcontrol oligo (mean ± SD; n = 2). *P < 0.05 using two-way ANOVA. (D) RT-qPCR of human SMN-FL mRNA relative to mouse gusb mRNA from the 5025 mousecortical neurons that were treated with 0.1, 0.3, 1.1, 3.3, or 10 μM EZH2 gapmer for 14 d (mean ± SD; n = 2). (E) RT-qPCR results of human SMN-FL mRNA fromSMA mouse from 5025WT SMA mouse model cortical neurons that were treated with a fixed concentration of RN-0027 (10 μM) in combination with in-creasing concentrations of a SCO for 14 d (mean ± SD; n = 2). (F) Human SMN protein levels from 5025 SMA mouse cortical neurons that were treated with afixed RN-0027 concentration in combination with increasing concentrations of a SCO for 14 d (mean ± SD; n = 2), as measured by ELISA.

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To assess the activity of RN-0005 in disease-relevant cells, weexamined SMN expression in two neuronal cell types. First, wegenerated induced pluripotent stem cells (iPSCs) derived fromSMA patient fibroblasts and differentiated them into SMI32+

motor neurons (SI Appendix, Fig. S6). After treating with RN-0005for 14 d, SMN-FL mRNA displayed a statistically significanttwofold increase relative to untreated or control-treated motorneurons (Fig. 5A). Both an inactive oligo that targets SMN-AS1but does not up-regulate SMN mRNA and an unrelated oligo thatdoes not target SMN-AS1 showed no effect on SMNmRNA levels.As expected, EZH2 knockdown also led to a similar increase inSMN-FL mRNA. We performed a time-course experiment withRN-0005 and observed a delayed increase in SMN-FL mRNAlevels in neurons relative to what was seen in fibroblasts. This maybe partially due to the mode of delivery (unassisted delivery versustransfection) and/or the nonproliferating state of the neuronal cellsversus the highly proliferative fibroblasts. Supporting the latter,the rate of H3K27me3 removal from chromatin of nondividingcells is slower than in proliferating cells (27). Taken together,these data show that disrupting the PRC2:SMN-AS1 interactionleads to SMN up-regulation in disease-relevant and postmitoticmotor neuronal cultures.We also prepared primary cortical neuronal cells from E14

embryos of the 5025 SMA mice and treated them with a chemicalvariant of RN-0005 that targets the same SMN-AS1 sequence andshows similar blocking of PRC2:SMN-AS1 and mRNA up-regu-lation (SI Appendix, Fig. S7) but has a more favorable in vivo safetyprofile. RN-0027 was added to the neurons for 14 d without ob-vious toxicity or changes in cell morphology (Fig. 5B). We observeda concentration-dependent increase in SMN-FL mRNA with athreefold increase at 10 μM following 14 d of treatment (Fig. 5C)in multiple experiments. Furthermore, the addition of a controloligo did not result in changes in SMN-FL levels. In agreementwith the results obtained from patient fibroblasts (Fig. 1B) andmotor neuron cultures (Fig. 5A), ex vivo cortical neurons treatedwith an EZH2 gapmer ASO displayed a concentration-dependentincrease in SMN-FL mRNA levels (Fig. 5D). Our findings from exvivo cortical neurons suggest that there is in vivo relevance of thismechanism in terminally differentiated neuronal cells.

Combination of Transcriptional Up-Regulation and SMN Exon 7 SpliceCorrection Increases SMN-FL mRNA. Splice-correcting modifiershave been designed to facilitate the inclusion of exon 7 duringSMN2 transcription (28), resulting in the production of SMN-FLmRNA and functional SMN protein. Although steady-state totalSMN mRNA levels would not increase with a splice-correctingmodifier, the shift to increase SMN-FL mRNA levels has beendemonstrated to be beneficial to survival in mice (29, 30) and inhumans (31). Because the transcriptional activation approach up-regulates SMN through a distinct mechanism from that of a splicecorrector, we reason that combining these two mechanisms will bemore effective than with a single approach. To this end, the 5025mouse cortical neurons, which only harbored human SMN2 andnot human SMN1, were treated with either a SCO, our transcrip-tional activating mixmer, or a combination of the two oligos for14 d to measure the levels of SMN-FL mRNA (Fig. 5E). Althoughtreatment with the SCO alone resulted in a 2–3-fold increase inSMN-FL mRNA, an additional 1.8-fold increase was observed incombination with the mixmer treatment. This additive effect wasalso detected with an increase in the human SMN protein levels bya human-specific ELISA (Fig. 5F). Although the SCO up-regulatedSMN protein levels ∼2.5-fold, the combination resulted in afourfold increase of SMN levels. These data further provide evi-dence that SMN-AS1 inhibition increases SMN-FL mRNA andSMN protein levels by a mechanism that is independent andcomplementary to that of splice correction. The increased SMN-FLmRNA and protein resulting from the combination approach mayprovide greater benefit in treating SMA.

DiscussionTreatments for SMA are focused on addressing symptomsranging from respiratory complications to muscle atrophy. Variousapproaches to treat SMA are being tested in clinical trials to ad-dress both neurological and muscular decline (reviewed by ref. 32).Splice-correcting therapies use ASOs or small molecules to pro-mote exon 7 inclusion. Recently, an ASO which includes exon 7 bythe splice-correcting approach, Spinraza, was approved by theFDA. Gene therapy replacement of SMN1 offers an alternativestrategy to increase levels of SMN protein and is currently beingtested. A neuroprotective agent may offer some resilience to motorneurons, but these agents have not proven effective in other neu-rodegenerative disorders such as ALS. A skeletal muscle enhanceris being evaluated to determine whether protecting the muscle willlessen disease progression.We report a transcriptional up-regulation method to selec-

tively up-regulate endogenous SMN mRNA and protein with theidentification and characterization of an lncRNA associated withthe SMN1 and SMN2 loci. The two genes are nearly identical insequence, resulting from a chromosomal duplication, which wouldsuggest that their regulation might be the same. We showed that inboth SMN1- and SMN2-mutated cell lines, SMN-AS1 is expressed.Moreover, SMN2 copy number in patient cell lines correlated withthe relative expression level of SMN-AS1, suggesting that thislncRNA is tightly associated with each copy of the gene. Takentogether, it is likely that SMN-AS1 regulation of expression of bothgenes might be similar. It remains possible that other mecha-nisms may contribute to regulating SMN1 and SMN2 expressiondifferentially, perhaps temporally or spatially, but we are unableto distinguish between them with our sequenced-based assays.Therefore, we address the two loci as one.Disruption of the lncRNA:PRC2 interaction resulted in

changes to SMN expression but not to expression changes in theneighboring genes based on our RNA-seq data, suggesting thatthe lncRNA functions in cis. The overall changes in PRC2 andRNA polymerase II occupancy and histone modifications suggestthat the increase in steady-state levels of SMN2 arises at thetranscriptional level in disease-relevant cell types. Indeed, whenmouse primary cortical neurons carrying copies of human SMN2were treated with our transcription-activating mixmers and aSCO, we observed an additive effect of increased SMN2 ex-pression beyond that offered by a splice-correcting therapy aloneto potentially confer greater therapeutic benefit.LncRNAs isolated by nuclear fractionation were shown to be

tethered to neighboring protein-coding genes (33). LncRNAshave diverse cellular functions and are critical for maintainingcellular identity (34). Furthermore, it has been demonstratedthat PRC2 is associated with lncRNAs, and it has been suggestedthat this relationship may serve to recruit PRC2 to specific sites(17, 35). In vitro studies and a recent study using iCLIP for PRC2have demonstrated that PRC2 interacts with RNAs nonspecificallyand that nascent transcripts may divert PRC2 from being recruitedto an actively transcribed gene (36). However, this does not ex-clude the possibility that lncRNAs may also interact with PRC2through specific interactions. Our data demonstrate transcriptionalup-regulation of SMN resulting from the loss of PRC2 associationwith the chromatin by targeting an oligo to disrupt a specificlncRNA: PRC2 interaction site. We believe that this interaction isspecific as well, as the disruption of SMN-AS1 with PRC2 does notchange the association of other lncRNAs with PRC2, and we seefewer pathway and expression changes compared with directlyknocking down PRC2 to increase SMN expression.In summary, we have demonstrated proof-of-concept that our

gene up-regulation technology disrupts the interaction betweenPRC2 and a lncRNA, which leads to the increased expression ofits associated protein-coding gene. Our approach of preventingPRC2 recruitment to a specific genomic location potentially

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offers greater selectivity and elicits fewer unintended side effectsthan small-molecule EZH1/2 inhibitors. Notably, this technologyachieves the degree of SMN up-regulation considered to betherapeutic for SMA. With this proof-of-concept, we believe thatour up-regulation platform could be applied to many other dis-eases in which a desirable gene is epigenetically silenced by atranscriptional repressive complex.

Experimental ProceduresRNA Sequencing. RNA from GM09677 fibroblasts that were transfected withRN-0005, SUZ12 gapmer ASO, and lipid controls were sequenced (300 bppaired-end) on the NextSeq500 using Illumina TruSeq stranded total RNA-seqlibrary preparation kits. Refer to SI Appendix, SI Experimental Procedures fora detailed explanation of the differential gene expression analyses.

Northern Blots.RNA preparation. Total RNA from human fetal brain and lung tissue wasobtained from Clontech and treated with RiboMinus (Life Technologies). Wefractionated 500 ng of rRNA-depleted RNA on a 1% agarose gel in 1× Mopsbuffer. RNA was capillary-transferred to BrightStar Plus nylon membrane(Ambion) overnight in 20× SSC buffer, then cross-linked by UV exposure. Formouse Northern blots, RNA was isolated from 5025 WT brain tissue and WTbrain tissue and treated with RiboMinus as above. Approximately 750 ngRNA was loaded per lane.Probe preparation. DNA templates containing a T7 promoter for in vitrosynthesis of radiolabeled RNA probes were generated by PCR from a humanfetal brain cDNA library or mouse brain cDNA library with primer pairs listedin SI Appendix, Table S1.

Stellaris RNA-FISH. Probe sets were designed against genomic regions listed inSI Appendix, Table S1. They were labeled with Quasar 570 (SMN1/2 exons),Quasar 670 (SMN1/2 introns), and Cal Fluor Red 610 (SMN1/2-AS1). StellarisRNA-FISH was performed as described in the Alternative Protocol for Ad-herent Cells (UI-207267 Rev. 1.0) with the following modifications: 12-mmdiameter coverslips were used. We used 25 μL hybridization solution with afinal concentration of each probe set of 250 nM. The wash buffer volumeswere halved. The FITC, Cy3, Cy3.5, and Cy5.5 channels were used to capturethe signals from each probe set, and the FITC channel was used to identifycellular autofluorescence. The filter sets from Chroma were 49001-ET-FITC,SP102v1-Cy3, SP103v2-Cy3.5, and 41023-Cy5.5. The exposure times were 1 s forFITC, Quasar 570, and Cal Fluor Red 610 and 2 s for Quasar 670. GM09677Human Eye Lens Fibroblast (Coriell) adherent cells were grown in Eagle’sMinimum Essential Medium (EMEM) (ATCC) in a humidified 37 °C incubator at5% CO2 in ambient air. F-12K and EMEM media were supplemented with10% (vol/vol) FBS (Fisher Product number SH30071.03) and 5 mL of Pen/Strep (Life Technologies). F-12 was further supplemented with Normocin(InvivoGen). Cells were grown on 12-mm microscope circular cover glassNo. 1 (Fisher #12–545-80) in 24-well flat-bottom cell culture plates (E&K).SMA fibroblasts were transfected at 70% confluence by using oligonucleo-tides complexed with Invitrogen Lipofectamine 3000 (Pub Part #100022234,Pub #MAN0009872, Rev. B.0) and fixed after 2 d. We used 2 ng DNA and 4 μLP3000 reagent per 50 μL of DNA master mix. We used 0.375 μL Lipofectamine3000 reagent per 25 μL of Opti-MEM.

RT-qPCR. Total RNA from 20 human tissues (Clontech) were used for cDNAsynthesis using High Capacity cDNA Reverse Transcription Kit (Applied Bio-systems). Data of RT-qPCR SMN-AS1 levels were normalized to levels from theadrenal gland. GM09677 fibroblasts were plated on a 24-well tissue cultureplate at 4 × 104 cells per well in MEM containing 10% (vol/vol) FBS and 1×nonessential amino acids. Fibroblasts were treated with oligos the followingday. After 2 d, cells were lysed and mRNA was purified using E-Z 96 Total RNAKit (Omega Bio-Tek). SMA iPS-derived motor neurons were lysed withTRIzol for RNA isolation per the manufacturer’s protocol. RNA from mousecortical neurons was extracted using the RNeasy kit (Qiagen) per themanufacturer’s protocol. All cDNAs were synthesized using High CapacitycDNA Reverse Transcription Kit (Applied Biosystems). SMN-FL, SMN Δ7,SMN Exon 1–2 and GUSB mRNA expression was quantified by predesignedTaqMan real-time PCR assays. A list of custom-designed real-time PCR assays islisted in SI Appendix, Table S1.

Oligonucleotide Transfection. SMA fibroblasts were transfected at 70% con-fluence by using oligonucleotides complexed with Lipofectamine 2000 (Invi-trogen) following the protocol suggested by the manufacturer in the 96-welland 24-well format. For ChIP, cells were transfected in 15-cm plates and were

transfected at 30 nMwith Lipofectamine 2000 at a final volume of 20 mL. Cellswere harvested 3 d posttransfection.

RIP. RIP was performed using the Magna RIP RNA-Binding Protein Immu-noprecipitation Kit (EMD Millipore) using ChIP-grade anti-SUZ12 (Abcam),anti-EZH2 (Abcam), and anti-SETD2 (USBiological Life Sciences) antibodies.RNA was extracted with TRIzol (Life Technologies) and transcribed to cDNAusing the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).qPCR was performed on a StepOnePlus Real Time PCR System (AppliedBiosystems) using TaqMan Fast Advanced Master mix (Applied Biosystems).

EMSA. DNA templates for EMSA probes containing T7 promoter sequenceswere generated by PCR using Phusion High Fidelity DNA Polymerase (NEB).The specific primer sequences are listed in SI Appendix, Table S1. EMSAs wereperformed as described previously (Cifuentes-Rojas et al., 2014) (22). RNAprobes were transcribed using the AmpliScribe T7 Flash Transcription Kit(Epicentre) and PAGE purified from 6% (vol/vol) TBE urea gel. RNA probeswere then dephosphorylated by calf intestinal alkaline phosphatase (NEB),purified by phenol-chloroform extraction, 5′ end-labeled with T4 PolynucleotideKinase (NEB) and [γ-32P] ATP (Perkin-Elmer), and purified with Illustra MicroSpinG-50 columns (GE Life Sciences). RNA probes were folded in 10 mM Tris pH 8.0,1 mM EDTA, and 300 mM NaCl by heating to 95 °C, followed by incubationsat 37 °C and at room temperature for 10 min each. MgCl2 and Hepes pH 7.5were then added to 10 mM each, and probes were put on ice. We mixed 1 μLof 2,000 cpm/mL (2 nM final concentration) folded RNA with PRC2 (EZH2/SUZ12/EED; BPS Bioscience) at the indicated concentration and 50 ng/mL yeasttRNA (Ambion) in 20 μL final concentration of binding buffer [50 mM Tris·HClpH 8.0, 100 mMNaCl, 5 mMMgCl2, 10 mg/mL BSA, 0.05% Nonidet P-40, 1 mMDTT, 20 U RNaseOUT (Invitrogen), and 5% (vol/vol) glycerol]. Binding reactionswere incubated for 20 min at 30 °C and applied on a 0.4% hyperstrengthagarose (Sigma) gel in THEM buffer (66 mM Hepes, 34 mM Tris, 0.1 mMdisodium EDTA, and 10 mMMgCl2). Gels were run for 1 h at 130 V with bufferrecirculation at 4 °C, dried, and exposed to a phosphorimager screen. Screenswere scanned in a Storm 860 phosphorimager (Molecular Dynamics), and datawere quantified by Quantity One and normalized as described (37). KDs werecalculated with Graphpad Prism by fitting the data to a one site-specificbinding model.

Western Blot. Cells were lysed 5 d posttransfection using the extraction bufferfrom the SMN ELISA kit (Enzo) with Protease inhibitor mixture tablets(Roche). Total protein content was determined with the total BCA assay(Promega) for equal loading. Samples and Hi Mark prestained ladder (Invi-trogen) were run on a 4% (vol/vol) Bis–Tris gel, and proteins were transferredto nitrocellulose membrane. The membrane was incubated in blockingbuffer (Licor) overnight at 4 °C. The SMN antibody (BD catalog no. 610646),Alpha tubulin antibody (Abcam catalog no. ab125267), and secondary anti-mouse and anti-rabbit 800 (Licor) were used, and the membrane was scannedwith Odessey (Licor). Band intensities for SMN-FL protein and α-tubulin werequantified using Image Studio software.

ELISA.GM09677 fibroblasts were plated on a 24-well tissue culture plate at 4×104 cells per well in MEM containing 10% (vol/vol) FBS and 1× nonessentialamino acids. Fibroblasts were treated with oligonucleotides the following day.After 5 d, cells were lysed and protein was quantified with the SMN ELISA Kit(Enzo Life Sciences, Inc.) and normalized to total protein content as deter-mined by Micro BCA Protein Assay Kit (Thermo Scientific). For the human-specific ELISA used with the cortical neurons, a similar protocol was used.Briefly, cells were washed in cold PBS and lysed in RIPA buffer supplementedwith protease inhibitor Complete Tablets, mini EDTA-free EASYpack (Roche).Lysates were quantified by BCA, and ∼20–30 μg were used. A mouse mono-clonal anti-SMN antibodywas captured on high binding plates (Pierce) at 1 μg/mL;after blocking with BSA in PBS-0.05% Tween-20, lysates were incubated for2 h at room temperature; a rabbit polyclonal human SMN-specific anti-body at 1 μg/mL was used for detection, followed by HRP-goat anti-rabbit(Invitrogen). Signal was measured with SuperSignal ELISA PICO chemilu-minescent substrate (Thermo). Total GAPDH in the lysates was also quan-tified by ELISA (R&D Systems); SMN protein concentration was normalizedto total GAPDH content.

Cortical Neuron Isolation. Brains were isolated from E14 SMNΔ7 embryos andthe cortex was dissected with the MACS neuronal tissue dissociation kit(Miltenyi Biotec). The collected cortical neurons were plated at 0.5 × 106 cellsper well in Neurobasal media (ThermoFisher), B-27 supplement (Thermo-Fisher), and GlutaMax (ThermoFisher) in a 24-well plate coated with poly–D-lysine (Fisher). Cells were incubated at 37 °C, 5% CO2 for 4 d, allowing the

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cells to mature and networks to form before unassisted delivery of RN-0005.After 14 d, the cells were harvested for RNA isolation.

iPS Cell Culturing and Motor Neuron Differentiation. SMA patient and controlsubject dermal fibroblasts or lymphoblastoid cell lines (LCLs) were obtainedfrom the Coriell Institute forMedical Research. All of the cell lines and protocolsin the present study were carried out in accordance with the guidelines ap-proved by the Stem Cell Research Oversight Committee (SCRO) and InstitutionalReview Board (IRB) at the Cedars–Sinai Medical Center under the auspice IRB-SCRO Protocols Pro00032834 (iPSC Core Repository and Stem Cell Program) andPro00024839 and Pro00036896 (Sareen Stem Cell Program). The iPSCs weregrown to near confluence under normal maintenance conditions before thestart of the differentiation as per protocols described previously (38). Briefly,IPSCs were then gently lifted by Accutase treatment for 5 min at 37 °C. Wesubsequently placed 1.5–2.5 × 104 cells in each well of a 384-well plate in de-fined neural differentiation medium with dual-SMAD inhibition (39). After 2 d,neural aggregates were transferred to low adherence flasks. Subsequently,neural aggregates were plated onto laminin-coated six-well plates to inducerosette formation in media supplemented with 0.1 μM retinoic acid and 1 μMpuromorphine along with 20 ng/mL BDNF, 200 ng/mL ascorbic acid, 20 ng/mLGDNF, and 1 mM dbcAMP. Neural rosettes were isolated, and the purifiedrosettes were subsequently supplemented with 100 ng/mL of EGF and FGF.These neural aggregates, termed iPSC-derived motor neuron precursorspheres (iMPSs), were expanded over a 5-wk period. For terminal differ-entiation, iMPSs were disassociated with Accutase and then plated ontolaminin-coated plates over a 21-d period before harvest using the MNmaturation media consisting of Neurobasal supplemented with 1% N2,ascorbic acid (200 ng/mL), dibutyryl cyclic adenosine monophosphate (1 μM),

BDNF (10 ng/mL), and GDNF (10 ng/mL). RN-0005 treatments were carried outduring this terminal differentiation period. Antibodies used for immunocy-tochemistry were as follows: SSEA4 and SOX2 (Millipore); TRA-1–60, TRA-1–81,OCT4, and NANOG (Stemgent); TuJ1 (β3-tubulin) and Map2 a/b (Sigma); ISLET1(R&D Systems); and SMI32 (Covance).

ChIP. Cells were cross-linked with 1% formaldehyde for 10 min at roomtemperature and then quenched with glycine. Chromatin was prepared andsonicated (Covaris S200) to a size range of 300–500 bp. Antibodies for H3,H3K27me3, H3K36me3, EZH2, and RNA polymerase II Serine 2 (Abcam) andH3K4me3 (Millipore) were coupled to Protein G magnetic beads (NEB),washed, and then resuspended in IP blocking buffer. Chromatin lysates wereadded to the beads and immunoprecipitated overnight at 4 °C. Antibodiesagainst H3, H3K36me3, RNA polymerase II phosphoserine 2, H3K27me3, andEZH2 were obtained from Abcam, and H3K4m3 antibody was obtained fromMillipore. We used 10 μg of antibody per IP. IPs were washed, RNase A (Roche)treated, and Proteinase K treated (Roche), and the cross-links were reversed byincubation overnight at 65 °C. DNA was purified, precipitated, and resus-pended in nuclease-free water. Custom TaqMan probe sets were used to de-termine enrichment of DNA. Probes were designed using the custom designtool on the Life Technologies website. Primer sequences are listed in SI Ap-pendix, Table S1.

All RNA-sequencing data were deposited in the Gene Expression Omnibus(GEO) database under accession no. GSE83549.

ACKNOWLEDGMENTS. We thank Jeannie T. Lee, Andrey Sivachenko, andJonathan C. Cherry for critical discussions and Jonathan Cherry and BrianBettencourt for reading of the manuscript.

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