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. Author manuscript; available in PMC: 2018 Feb 6.
Published in final edited form as: Cell Rep. 2017 Aug 22;20(8):1950–1963. doi: 10.1016/j.celrep.2017.07.068

Regulation of peripheral myelination through transcriptional buffering of Egr2 by an antisense long noncoding RNA

Margot Martinez-Moreno 1,2, Timothy Mark O’Shea 3,4, John P Zepecki 1,2, Alexander Olaru 1, Jennifer K Ness 1, Robert Langer 3,4, Nikos Tapinos 1,2,*
PMCID: PMC5800313  NIHMSID: NIHMS909536  PMID: 28834756

Summary

Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA). During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of the Egr2-AS-RNA in DRG cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of the Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene-expression profile and significantly delays demyelination. The Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2 and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of the Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to the Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in PNS biology.

eTOC blurb

Martinez-Moreno et al., report a role for a long non-coding RNA antisense to the promoter of Egr2-AS-RNA, during the response to peripheral nerve injury. Inhibition of the Egr2-AS-RNA following sciatic nerve injury reverts EGR2-mediated gene expression and delays demyelination.

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Introduction

In the vertebrate peripheral nervous system (PNS), Schwann cells (SCs) produce the myelin sheath, the specialized membrane structure that allows rapid nerve conduction. In recent years, significant progress has been made in the identification of key transcriptional regulators of myelination. Evidence generated in the mouse suggests that transcription factor EGR2 plays the role of a central regulator in this process: (1) EGR2 is activated in SCs after axonal contact, before myelination (Murphy et al., 1996; Topilko et al., 1994); (2) Egr2 null or hypomorphic mutations result in blockade of SCs at the promyelinating stage, after the establishment of 1:1 ratio with the axons rendering them unable to proceed with the myelination process (Le et al., 2005; Topilko et al., 1994) (3) forced Egr2 expression in SCs results in the activation of genes encoding structural myelin proteins and enzymes involved in lipid synthesis (Nagarajan et al., 2001) and (4) downregulation of EGR2 expression after peripheral nerve injury results in demyelination (Ghislain et al., 2002; Zorick et al., 1996). In addition to the mouse studies, the association of various dominant or recessive Egr2 mutations with several types of human peripheral neuropathies supports the crucial role of Egr2 in the control of SC myelination (Bellone et al., 1999; Timmerman et al., 1999; Warner et al., 1998). Intracellular signaling pathways activated by both membrane-bound and soluble neuregulins regulate the expression of EGR2 in SCs (Murphy et al., 1996; Svaren and Meijer, 2008; Taveggia et al., 2005). Activation of the MEK-ERK1/2 cascade by neuregulin is responsible for the activation of the YY1 transcription factor, which binds to Egr2 promoter and regulates Egr2 expression (He et al., 2010). Ablation of ERK/12 signaling in Erk1/2CKO(Dhh) sciatic nerves leads to profound inhibition of EGR2 expression and severe hypomyelination (Newbern et al., 2011).

The aforementioned studies reflect the “classical” paradigm of transcriptional regulation where signaling intermediates activate transcription factors, which in turn bind specific DNA motifs located on promoters to regulate the expression of target genes. However, the role of epigenetic mechanisms (here taken to mean mechanisms (here taken to mean mechanisms such as histone modifications and ncRNAs that alter gene expression without changes in DNA sequence) orchestrated by non-coding RNAs that regulate transcription (Hawkins and Morris, 2008) have not been studied. In human cells there are two independent mechanisms that confer transcriptional gene silencing (TGS): i) a miRNA-directed mechanism and ii) a long-antisense RNA mechanism (Morris, 2009). Both short (miRNA) and long (antisense) RNA mediated TGS in human cells involves the interaction of RNA with promoter regions (Kim et al., 2008; Klase et al., 2007; Omoto and Fujii, 2005; Tan et al., 2009).

Here we describe a long non-coding RNA antisense to the proximal promoter of Egr2. The Egr2-AS-RNA shows increased expression during acute peripheral nerve injury. Expression of the Egr2-AS-RNA regulates the levels of Egr2 in SCs and in vivo inhibition of Egr2-AS-RNA results in the rescue of the EGR2–mediated gene expression profile and delay of demyelination following peripheral nerve injury. The Egr2-AS-RNA gradually recruits an epigenetic remodeling complex on the Egr2 promoter while, expression of the Egr2-AS-RNA is regulated by ERK1/2 signaling to YY1, which binds the Egr2-AS-RNA in the context of chromatin. Finally, YY1 mediates the interaction of the Egr2-AS-RNA with the chromatin remodeling factor ESH2 while, loss of Ser184 of YY1 induces direct binding of YY1 to the Egr2-AS-RNA.

Results

Discovery of an antisense long non-coding RNA at the 5′-UTR of Egr2

We have recently shown that miR-709 induces transcriptional gene silencing of Egr2 by binding to the MSE region of the Egr2 promoter (Adilakshmi et al., 2012). These data generated a hypothesis regarding the possible transcriptional regulation of the proximal promoter of Egr2 by antisense RNA. To determine if an antisense long non-coding RNA is present at the 5′-UTR of Egr2 we employed a modified 5′-RACE protocol to amplify the antisense strand. The resulting antisense product was cloned and the sequence is presented in Supplementary Figure S1A. Attempts to extend the RACE amplification further upstream using primer walking did not reveal any results (data not shown), which suggests that either the antisense RNA is ~1000nt long or that it is partially amplified with our RACE protocol. Next, we performed homology search using the rat antisense sequence against the mouse genome to identify degree of homology between rat and mouse. This showed 92% homology (Supplementary Figure S1B), which allowed us to design mouse-specific primers and perform a strand-specific RT-PCR to amplify the antisense product from total RNA isolated from mouse sciatic nerves. To determine if the AS-RNA has been identified before, we performed BLAST search using LNCipedia (Volders et al., 2013; Volders et al., 2015) and lncRNAdb (Amaral et al., 2011; Quek et al., 2015). The search returned no matches which suggests that this RNA is a previously undescribed long non-coding antisense RNA that we subsequently refer to as Egr2-AS-RNA. Finally, to independently validate the expression of the Egr2-AS-RNA, we examined publicly available RNAseq data from three mouse sciatic nerves to identify RNAseq reads that map to the identified AS-RNA transcript (Poitelon et al., 2016). We found that the number of Reads-per-Kilobase-per Million (RPKM) of the Egr2-AS-RNA range between 1.6 – 2.5 and are on average 5 times more than the median number of RPKM over the entire ~26K refseq transcripts (Supplementary table S1A) in sciatic nerves. In addition, the average relative abundance of the Egr2-AS-RNA in the three sciatic nerves was 43 times lower compared to the expression levels of the Egr2 transcript (Supplementary table S1B). Then we estimated the coordinates of the mature Egr2-AS-RNA and identified that the mature Egr2-AS-RNA sequence spans a 470-nucleotide region (Chr:10, 67537250–67537720) with almost perfect sequence fidelity (Supplementary Figures S2A & S2B). The discrepancy between the length of the mature AS-RNA (470 nt) and the sequence detected by the RACE protocol (~1000 nt) is attributed to the random decamers used for the amplification of the RACE product and the possibility that part of the Egr2-AS-RNA could be spliced after 5′-end capping.

The Egr2-AS-RNA is expressed in mouse sciatic nerves and is significantly increased following nerve injury

To determine if the Egr2-AS-RNA is expressed during the postnatal development of the mouse sciatic nerve we performed strand-specific qPCR using RNA isolated from P1, P5, P7 and 3 moth old mouse sciatic nerves. This showed that the Egr2-AS-RNA is expressed throughout these time intervals with the highest expression in P1 sciatic nerves (Figure 1A). To detect the expression and cell specificity of the Egr2-AS-RNA in mouse sciatic nerves we performed multiplex fluorescence in situ hybridization targeting the ERgr2-AS-RNA in combination with the mouse S100b, which was used as SC specific marker. We detected specific signal for the Egr2-AS-RNA in the cytoplasm (Figure 1B, arrowheads) and in the nucleus (Figure 1B, arrow) of S100b+ SCs. To examine if the Egr2-AS-RNA plays a role in the regulation of Egr2 expression following peripheral nerve injury, we performed strand-specific qPCR to detect the expression of the Egr2-AS-RNA and the Egr2 mRNA 6hrs, 12hrs, 24hrs, 2days, 5days and 7days following mouse sciatic nerve transection. We discovered that the expression of the Egr2-AS-RNA exhibits a statistically significant increase 6 and 12 hours after sciatic nerve injury (Figure 1C). The expression of the Egr2 mRNA shows statistically significant downregulation at 12 and 24 hours (Figure 1D) and rebounds in days 2, 5 and 7 when expression of the Egr2-AS-RNA is minimal. Finally, EGR2 protein expression in sciatic nerve lysates is significantly reduced by 48 hours after injury (Supplementary Figure S3A & S3B).

Figure 1. An Egr2 antisense RNA transcript is expressed during sciatic nerve development and is up-regulated after peripheral nerve injury.

Figure 1

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A) Expression of the Egr2-AS-RNA was detected by strand specific qPCR in P1, P5, P7 and 3month old mouse sciatic nerves. B) In situ hybridization in mouse sciatic nerves shows the expression of the Egr2-AS-RNA (green signal) in the cytoplasm (arrowheads) and nucleus (arrow) of S100b positive (red signal) SCs. Positive control shows expression of Cyclophilin B (PPIB) (green) and POLR2A (RNA polymerase II polypeptide) (red). Negative control shows no signal using a Bacterial dapB probe. Scale: 100μ. C) Expression of the Egr2-AS-RNA in non-injured sciatic nerves and in sciatic nerves 6hrs, 12hrs, 24hrs, 2days, 5days and 7days following sciatic nerve injury. The experiments were repeated three times and significance was calculated with One-Way ANOVA (F (6,22) = 22.19, p < 0.0001), followed by post-hoc Dunnett’s test: ** p<0.005, **** p<0.00005. D) Expression of Egr2 mRNA in non-injured sciatic nerves and in sciatic nerves 6hrs, 12hrs, 24hrs, 2days, 5days and 7days following sciatic nerve injury. The experiments were repeated three times and the results are presented as average ± standard deviation (s.d.). The experiments were repeated three times and significance was calculated with One-Way ANOVA (F (6,15) = 9.458, p < 0.0001), followed by post-hoc Dunnett’s test: *** p<0.0005. E) Absolute abundance of the Egr2-AS-RNA is significantly lower in control sciatic nerves compared to injured nerves as determined by One-Way ANOVA, F=6.935, p = 0.0161, using sciatic nerves from 3 mice per condition.

Quantification of absolute levels of the Egr2-AS-RNA in mouse sciatic nerves

In order to determine the absolute abundance of the Egr2-AS-RNA in mouse sciatic nerves, we prepared limiting dilutions (10ng -1pg) of a known amount of the Egr2-AS-RNA and generated a standard amplification curve of the dilutions using qPCR as described before (Lu and Tsourkas, 2009). The Ct values obtained from the amplification of the Egr2-AS-RNA were then projected on the standard curve to determine the concentration of the Egr2-AS-RNA per 100ng total RNA that was used as template in all qPCR reactions. This analysis showed that the average concentration of the Egr2-AS-RNA in non-injured sciatic nerves was 200pg/100ng RNA, while in injured nerves the average concentration fluctuated between 350 – 650 pg/100ng RNA depending on the time interval following nerve injury (Figure 1E). The low expression levels of the Egr2-AS-RNA are within the range of expression of lncRNAs in eukaryotic cells (Mortazavi et al., 2008; Palazzo and Lee, 2015; Ramskold et al., 2009). The fact that expression levels increase by an average of 4-fold following sciatic nerve injury implies specific functionality of the Egr2-AS-RNA in injured peripheral nerves.

Ectopic expression of the Egr2-AS-RNA results in demyelination and inhibition of Egr2 mRNA expression

Since increased Egr2-AS-RNA expression correlates with reduced Egr2 mRNA levels after sciatic nerve injury, we sought to demonstrate that the Egr2-AS-RNA could induce silencing of the Egr2 transcript expression. We generated a lentivirus expressing the Egr2-AS-RNA to infect mouse DRG explant cultures 14 days after the addition of ascorbic acid to ensure the presence of myelinated axons. We demonstrate that overexpression of the Egr2-AS-RNA (Figure 2E) results in statistically significant inhibition of Egr2 mRNA expression as detected by qPCR (Figure 2A). In addition, ectopic expression of the Egr2-AS-RNA results in statistically significant inhibition of EGR2 protein expression (Figure 2B). To examine the effect of the ectopic expression of Egr2-AS-RNA on myelination we infected myelinated mouse DRG cultures with pLenti-AS-RNA or pLenti-Ctrl. To show specificity of the Egr2-AS-RNA effect on myelination we incubated the pLenti-AS-RNA infected cultures with a scrambled oligonucleotide GapMer (scrambled complementary strand of the AS-RNA) or an Egr2-AS-RNA GapMer (complementary to the Egr2-AS-RNA sequence). One week after infection and treatment with the GapMers, we stained the cultures with Myelin Basic Protein (MBP) and Neurofilament (NF) antibodies to detect myelin internodes and integrity of the underlying axons respectively (Figure 2C). Infection with the pLenti-AS-RNA induces significant expression of Egr2-AS-RNA, which is not affected by the addition of scrambled GapMers, while Egr2-AS-RNA GapMers induce a significant reduction in the amount of Egr2-AS-RNA in the cultures (Figure 2E). Moreover, addition of the Egr2-AS-RNA GapMers rescues the demyelination phenotype observed in cultures infected with pLenti-AS-RNA while scrambled GapMers have no effect (Figure 2D & 2F). There was no difference in total cell numbers between the cultures (Supplementary Figure S3C).

Figure 2. Ectopic expression of the Egr2-AS-RNA inhibits the expression of Egr2 mRNA and induces demyelination.

Figure 2

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A) Lenti-AS-RNA infected cultures exhibit significant decrease in the expression of the Egr2 mRNA as compared to Lenti-control infected cultures. The experiment was repeated three times and significance was calculated with a two-tailed unpaired Student’s t-test and the results are presented as average ± s.d. (**p =0.0005, t=5.571, df=8). B) Representative Western blot showing expression of EGR2 after infection of DRG cultures with Lenti-Control or Lenti-AS-RNA. Actin was used as loading control. Densitometric analysis shows that ectopic expression of the Egr2-AS-RNA results in a statistically significant decrease of EGR2. The results are presented as average ± s.d. from n=5 per condition. Significance was calculated with two-tailed unpaired Student’s t-test (t=4.722, df=8, *p = 0.015). C) Representative images of Lenti-Control and Lenti-AS-RNA infected DRG cultures showing extensive demyelination in the cultures infected with Lenti-AS-RNA. Myelinated internodes were stained with MBP (red) and the nuclei stained with DAPI (Blue) (Upper panels). Lower left panel shows higher magnification of the myelin internodes (red stained with MBP) and the axons (green stained with neurofilament) in Lenti-control infected cultures. In Lenti-AS-RNA infected cultures we detect myelin debris (stained red with MBP) while the axons retain their integrity expressing NF (green). Scale bars: 100μ. D) Rescue of AS-RNA induced demyelination with oligonucleotide inhibitors of the Egr2-AS-RNA (GapMers). E) Lenti-AS-RNA infected cultures exhibit significant increase in the expression of the AS-RNA as compared to Lenti-control infected cultures and the expression of the AS-RNA is not affected by the addition of scrambled GapMers (Two-tailed, unpaired Student’s t-test, p = 0.0135, t=3.458 df=6). Treatment of the Lenti-AS-RNA infected DRG cultures with Egr2-AS-RNA specific GapMers results in significant inhibition of Egr2-AS-RNA expression as compared to Lenti-AS-RNA infected cultures treated with scrambled GapMers (Two-tailed, unpaired Student’s t-test, p=0.0001, t=14.04 df=6). F) Myelin internodes from ten 10X fields per culture from 4 cultures per condition were measured and the results are presented as average ± s.d. Significance was calculated with a two-tailed unpaired Student’s t-test. Lenti-AS-RNA infected cultures treated with a control (scrambled) GapMer have significantly fewer myelinated internodes compared to Lenti-Control infected cultures (p = 0.0007, t=5.352 df=8). An Egr2-AS-RNA specific GapMer rescues the Lenti-AS-RNA infected cultures from the AS-RNA induced demyelination (p < 0.0001, t=7.949 df=70).

Inhibition of the Egr2-AS-RNA expression using oligonucleotide GapMers results in delay of demyelination following peripheral nerve injury

We developed an in situ, non-swelling, biodegradable hydrogel (O’Shea et al., 2015) loaded with oligonucleotide GapMers (20-mers) against the Egr2-AS-RNA. The GapMer-infused hydrogel was applied to the sciatic nerve at the time of transection. We designed five GapMers targeting different areas of the Egr2-AS-RNA and a scrambled GapMer for control. Four GapMers induced significant inhibition of the Egr2-AS-RNA expression as compared to transected nerves that received hydrogel only or hydrogel + scrambled GapMers (Figure 3A). Inhibition of Egr2-AS-RNA expression with each of these GapMers results in statistically significant increase in the expression of Egr2 mRNA following peripheral nerve injury (Figure 3B). Using Electron Microscopy, we discovered that addition of the hydrogel+GapMer at the time of sciatic nerve transection delays the injury-induced demyelination at days 2, 5 and 7 after nerve injury compared to injured nerves alone or injured nerves treated with hydrogel+scrambled GapMer (Figure 3C). Sub-therapeutic concentrations of GapMer remained within the hydrogel beyond 7 days, consistent with in vitro release results for biomacromolecules of a similar molecular weight (O’Shea et al., 2015) and consequently the study was not extended further. Inhibition of the Egr2-AS-RNA expression using GapMers results in significant reduction in the percentage of demyelinated fibers and increase in the percentage of myelinated fibers 2, 5 and 7 days following complete sciatic nerve transection (Figure 3D). A statistically significant increase in the total number of myelinated fibers 5 and 7 days following complete sciatic nerve transection was also observed (Figure 3E).

Figure 3. In vivo inhibition of the Egr2-AS-RNA expression results in delayed demyelination after sciatic nerve injury.

Figure 3

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A) Effect of five separate oligonucleotide GapMers complementary to five different sequence motifs of the Egr2-AS-RNA on the expression of the Egr2-AS-RNA in mouse sciatic nerves. Quantification was performed with qPCR combining RNA from 3 separate mice per individual GapMer. Four GapMers induced significant inhibition of the AS-RNA expression (One-Way ANOVA (5,13) = 5.846, p = 0.0111) followed by a post-hoc Dunnett’s test (* p < 0.05) as compared to transected nerves that received Hydrogel only (Lesion) or hydrogel + scrambled GapMers. B) Inhibition of AS-RNA with each of these GapMers results in significant increase in the expression of Egr2 mRNA as compared to non-treated or scrambled-GapMer treated injured sciatic nerves (One-Way ANOVA (F(5,7) = 7.175, p = 0.0111) followed by post-hoc Dunnett (* p < 0.05), 3 sciatic nerves per condition). C) Nerves that received Hydrogel only or Hydrogel+Scrambled GapMers show varying degrees of demyelination and axonal damage at 2, 5 and 7 days after sciatic nerve transection. Animals treated with Hydrogel+GapMer appear to have less demyelination, less axonal degeneration and the endoneural space appears more compact without extensive collagen depositions, which is more evident at day 7 as compared to animals treated with Hydrogel only or Hydrogel+Scrambled GapMers. D) Quantification of the myelinated and demyelinated fibers as a percentage of the total number of fibers in non-treated, hydrogel+scrambled GapMer treated and hydrogel+GapMer treated nerves following sciatic nerve injury. Inhibition of the Egr2-AS-RNA expression using specific oligonucleotide GapMers results in significant reduction in the percentage of demyelinated fibers and increase in the percentage of myelinated fibers 2, 5 and 7 days following complete sciatic nerve transection (*:p<0.05, two-tailed Student’s t-test of unpaired samples). E) Quantification of the total number of myelinated axons in 15 random semithin sections from an area extending 0.5mm to 5mm distal to the sciatic nerve transection. Animals treated with hydrogel+AS-RNA GapMers have significantly higher number of myelinated fibers 2 and 5 days following complete sciatic nerve transection as compared to untreated animals or animals treated with hydrogel+scrambled GapMers (*:p<0.05, two-tailed Student’s t-test of unpaired samples).

In vivo inhibition of Egr2-AS-RNA expression rescues the EGR2-mediated gene expression profile following peripheral nerve injury

As shown above (Figure 1C), peripheral nerve injury results in an acute and significant increase in Egr2-AS-RNA expression up to 12hrs post-injury. To determine how the Egr2-AS-RNA affects the expression of EGR2 regulated transcripts following sciatic nerve injury, we treated sciatic nerves with hydrogel+GapMer to inhibit the expression of the Egr2-AS-RNA at the time of sciatic nerve transection and up to 5 days post-injury. This showed that inhibition of Egr2-AS-RNA expression in fully transected nerves rescues the inhibition of Egr2 transcript expression (Figure 4A) and either rescues or delays the inhibition of EGR2-regulated genes that encode structural myelin proteins or transcription factors (Figure 4A). To determine if the delayed inhibition of gene expression results in delayed downregulation of PMP22 and MPZ myelin protein expression we analyzed sciatic nerve lysates 2, 5, and 7 days post injury treated with hydrogel+GapMer, hydrogel+Scrambled GapMer and not treated injured nerves. We show that inhibition of Egr2-AS-RNA expression results in significant preservation of PMP22 and MPZ protein expression 7 days following complete sciatic nerve transection (Figure 4B–C). The delayed inhibition of structural myelin proteins correlates with the delay in demyelination observed after inhibition of the Egr2-AS-RNA expression (Figure 3C–E).

Figure 4. The Egr2-AS-RNA regulates Egr2 gene and Egr2 target gene expression during nerve injury response.

Figure 4

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A) Volcano plots of Log2 fold change for EGR2 regulated genes in non-injured vs injured sciatic nerves, and injured vs injured treated with hydrogel + GapMers nerves. The x-axis shows the Log2 of the fold change (FC) between the conditions. Vertical central line represents no difference in expression and the area between the two equidistant lines in both sides of the central line includes genes showing non-significant change of expression (black dots). At the left side of the lines, the genes with negative FC are depicted (decreased expression, blue dots), while at the right side of the lines the genes with positive FC (increased expression) are shown (yellow dots). The y-axis shows the –log of the p-value, which means that genes with low p-value (more significant) appear towards the top of the plot. Horizontal line splits the significant results (p<0.005, above) from the non-significant (below). B) Representative results from three independent experiments using two sciatic nerve isolates 2, 5 and 7 days post-injury showing expression of PMP22 and MPZ. Actin was used as loading control. C) Densitometric analysis of the Western blot results shows that 7 days after sciatic nerve injury PMP22 and MPZ exhibit significantly higher expression in animals treated with hydrogel + AS-RNA GapMers as compared to injured animals or injured animals with hydrogel + Scrambled GapMers. The results are normalized to the expression of Actin and presented as average ± standard deviation from three independent experiments. Significance was calculated with One-Way ANOVA (p < 0.001, F (3, 4) = 25.87 for PMP22, p = 0.0019, F (2,6)=21.3 for MPZ) followed by post-hoc Dunnett (** p < 0.005).

The Egr2-AS-RNA inhibits nascent transcription of Egr2 and mediates a gradual recruitment of a chromatin remodeling complex on the Egr2 promoter

In order to distinguish if the effect of the Egr2-AS-RNA on Egr2 expression is due to a direct effect on transcription, we performed nuclear run-on assays in SCs infected with Lenti-AS-RNA or Lenti-Control. We show that expression of the Egr2-AS-RNA results in statistically significant inhibition of nascent transcription of Egr2 (Figure 5A). To determine if the Egr2-AS-RNA participates in the formation of a chromatin remodeling complex on the Egr2 promoter we performed ChIP using sciatic nerve chromatin with antibodies against EZH2, AGO1, AGO2, or the tri-methylated histone 3 (H3K27Me3) followed by qPCR to detect the presence of the Egr2 promoter and the Egr2-AS-RNA in the complex. This revealed that 48 hours after sciatic nerve injury EZH2, AGO1, AGO2 and H3K27me3 are localized on the Egr2 promoter, and this interaction is inhibited after treatment with RNAse H, which means that the interaction requires the presence of an RNA-DNA hybrid (Figure 5B). Next, we asked if the Egr2-AS-RNA is the responsible RNA species for the recruitment of the remodeling complex on the Egr2 promoter following sciatic nerve injury. We performed ChIP using chromatin from injured sciatic nerves 6, 24 and 48 hours after injury. To identify the effect of the Egr2-AS-RNA on the recruitment of EZH2, AGO1, AGO2 and H3K27me3 on Egr2 promoter we inhibited the expression of the Egr2-AS-RNA with the addition of hydrogel + GapMer, while control nerves were treated with hydrogel + scrambled GapMer. We show that 6 hours post-injury only AGO1 and AGO2 are present on Egr2 promoter and inhibition of Egr2-AS-RNA expression inhibits their recruitment on the promoter (Figure 5C). At 24hrs post-injury AGO2 and H3K27me3 are present and inhibition of the Egr2-AS-RNA expression abolishes their recruitment on the Egr2 promoter (Figure 5D). At 48hrs post-injury EZH2, AGO1, AGO2 and H3K27me3 are all recruited on Egr2 promoter and this depends on the presence of the Egr2-AS-RNA since inhibition of its expression results in inhibition of EZH2, AGO1, AGO2 and H3K27me3 binding on the Egr2 promoter (Figure 5E). To discover if the gradual recruitment of the repressive complex by the Egr2-AS-RNA mediates transcriptional repression of Egr2 mRNA we compared the ChIP results with the expression of Egr2 mRNA at 6, 24 and 48 hours following sciatic nerve injury. We show that 6hrs post-injury where the Egr2-AS-RNA recruits AGO1 and AGO2 on Egr2 promoter (Figure 5C), Egr2 transcription is repressed and inhibition of the Egr2-AS-RNA with specific GapMers induces significant 38-fold increase in Egr2 expression compared to injured nerves treated with the scrambled GapMers (Figure 5F-6hrs). At 24hrs post-injury the Egr2-AS-RNA mediated recruitment of AGO2 and H3K27me3 on Egr2 promoter (Figure 5D) does not correlate with Egr2 transcriptional repression since the levels of Egr2 transcript are equal between injured nerves treated with the AS-RNA GapMers or the Scrambled GapMers (Figure 5F-24hrs). Finally, at 48hrs post-injury the Egr2-AS-RNA-mediated recruitment of EZH2, AGO1, AGO2 and H3K27me3 on Egr2 promoter (Figure 5E) correlates with a modest but significant transcriptional repression of Egr2 since inhibition of Egr2-AS-RNA expression with GapMers induces a 3-fold increase in Egr2 transcript levels compared to injured nerves treated with the scrambled GapMers (Figure 5F-48hrs).

Figure 5. The Egr2-AS-RNA mediates gradual recruitment of a remodeling complex on Egr2 promoter.

Figure 5

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A) Expression of the Egr2-AS-RNA induces a statistically significant decrease in nascent transcription of Egr2 as quantified by qPCR. (N = 8 in control cells, N=9 in Egr2-AS-RNA cells, 3 independent experiments, p = 0.0012, t=3.967, df=15). B) In vivo ChIP using sciatic nerve chromatin with antibodies against EZH2, AGO1, AGO2 and H3K27Me3 with or without RNAse H. Graph shows the average ± standard deviation from three independent experiments using sciatic nerves from three animals per experiment. Normalization was performed with values acquired from ChIPs with isotype matched IgG. Significance was calculated with unpaired two-tailed Student’s t-test, p = 0.040, t=4.836, df=2 for EZH2; p = 0.044, t=2.902, df=4 for AGO1; p = 0.045, t=4.514, df=2 for AGO2; p = 0.0012, t=4.348, df=4 for H3K27. C–E) In vivo DNA ChIP using chromatin from injured sciatic nerves 6, 24 and 48 hours after injury. At 6hrs post-injury only AGO1 and AGO2 are present on Egr2 promoter and inhibition of Egr2-AS-RNA expression inhibits their recruitment on the promoter (3 independent experiments; unpaired two-tailed Student’s t-test, p = 0.012, t=5.710, df=4 for AGO1; p = 0.023, t=2.105, df=4 for AGO2) (C). At 24hrs post-injury AGO2 and H3K27me3 are present and inhibition of the AS-RNA expression abolishes their recruitment on the Egr2 promoter (3 independent experiments; unpaired two-tailed Student’s t-test, p = 0.029, t=2.832, df=6 for AGO2; p = 0.049, t=2.232, df=10 for H3K27me3) (D). At 48hrs post-injury EZH2, AGO1, AGO2 and H3K27me3 are all recruited on Egr2 promoter and this depends on the presence of the Egr2-AS-RNA since inhibition of the AS-RNA expression results in inhibition of EZH2, AGO1, AGO2 and H3K27me3 binding on the Egr2 promoter (3 independent experiments; unpaired two-tailed Student’s t-test, p = 0.033, t=3.188, df=4 for EZH2; p = 0.017, t=3.921, df=4 for AGO1; p = 0.018, t=3.826, df=4 for AGO2; p = 0.035, t=2.714, df=6 for H3K27me3) (E). F) Transcript expression of Egr2 6hrs, 24hrs and 48hrs post-injury using RNA from injured sciatic nerves treated with AS-RNA GapMers or Scrambled Gapmers. The results are presented as fold change of Egr2 transcript expression in AS-RNA GapMer treated nerves versus Scrambled GapMer treated nerves. The experiment was repeated three times and the data are presented as average± standard deviation. Fold change above 2 was set arbitrarily and is used in conjunction with p<0.05 to determine significant differences in gene expression.

Expression of the Egr2-AS-RNA is regulated by ERK1/2 signaling

Since the Egr2-AS-RNA has a direct effect on the expression of Egr2 transcript and protein levels (Figures 1–4 & Figure S1) we hypothesized that neuregulin-mediated ERK1/2 signaling could affect the expression of the Egr2-AS-RNA in SCs since it has also been shown to affect the expression levels of Egr2 (Newbern et al., 2011). Inhibition of neuregulin-induced ERK1/2 activation using UO126 in SCs, results in significant inhibition of ERK1/2 phosphorylation, significant reduction of EGR2 protein levels (Figure 6A & B), significant up-regulation of the Egr2-AS-RNA expression and inhibition of Egr2 transcript expression (Figure 6C). Our data suggest that neuregulin-mediated ERK1/2 signaling exerts negative regulation on the Egr2-AS-RNA expression in SCs. Recently, it was shown that loss of axonal contact causes SCs to induce NRG1 Type I expression through a MAPK-dependent pathway (Stassart et al., 2013). We determined if NRG1 Type III or Type I exert a distinct role on the expression of the Egr2-AS-RNA. We show that stimulation of SCs with NRG1 Type III is the main signal that exerts an acute negative regulation on the expression of the Egr2-AS-RNA (Supplementary Figure S4). NRG1 Type I did not have any effect after 3hrs but showed a gradual inhibition of Egr2-AS-RNA expression in 6hrs and 24hrs. Our data could explain the acute increase in the expression of the Egr2-AS-RNA immediately after nerve injury (Figure 1C) while the gradual inhibition of Egr2-AS-RNA expression after 24hrs post-injury (Figure 1C) may reflect gradual inhibition by NRG1 Type I as SCs induce its expression.

Figure 6. Role of NRG1-ERK1/2 signaling and YY1 in the regulation of the Egr2-AS-RNA expression.

Figure 6

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A) Inhibition of NRG1-induced Erk1/2 phosphorylation in SCs using UO126 results in inhibition of phopsho-ERK1/2 expression and loss of EGR2 expression. Total ERK1/2 and Actin were used as loading controls. B) Densitometric quantification of inhibition of ERK1/2 phosphorylation and EGR2 expression following incubation of SCs with UO126. Results are presented as average ± standard deviation from three independent experiments using two separate protein isolations per experiment per condition. (p < 0.0001, t=8.99, df=14 for p-ERK1/2; p = 0.0054, t=4.24, df=6 for EGR2). C) qPCR for the Egr2-AS-RNA and the Egr2 mRNA following inhibition of ERK1/2 signaling with UO126 in SCs shows significant increase of the Egr2-AS-RNA levels and inhibition of Egr2 mRNA expression. (n= 3 independent experiments. Unpaired 2-tailed Student’s t-test. p = 0.044, t=2.44, df=7 for Egr2-AS-RNA; p = 0.017, t=4.32, df=5 for Egr2). D) Schematic showing the position of the S1 site upstream of the Egr2 transcription start site (TSS) and flanking the 5′- of the AS-RNA. This site (~100nt) contains a YY1 binding motif and a TATA box (red sequence) in both sense and antisense orientation. MSE stands for the Myelin Specific Element, which is located at the 3′-UTR of the Egr2 gene. E) YY1 DNA-ChIP using chromatin isolated from SCs that were untreated or treated with UO126 in the presence of NRG1 overnight. Association of YY1 with the S1 and S2 regions of the Egr2 promoter was examined with qPCR. YY1 associates only with the S2 region of the Egr2 promoter and the association is partially inhibited after treatment with UO126. The experiments were repeated three times and the results are presented as average± standard deviation. Significance was calculated with a Student’s t-test (p=0.0012, t=28.96, df=12). F) RNA immunoprecipitation (RIP) using RNA isolated from SCs that were untreated or treated with UO126 in the presence of NRG1 overnight. Association of YY1 with the S1 and S2 regions of AS-RNA was examined with qPCR. The experiments were repeated three times and the results are presented as average± standard deviation. Significance was calculated with a Student’s t-test (p = 0.0085, t=3.842, df=6 for S2; p = 0.0362, t=3.099, df=4 for S1). G) SCs transfected with YY1 siRNA for 48hrs show inhibition of YY1 protein expression as compared to SCs transfected with non-targeting siRNAs. Actin was used as loading control. H) RIP with antibodies against EZH2, AGO1, AGO2 and H3K27me3 using RNA from YY1 siRNA or non-targeting siRNA transfected SCs. Results are presented as average ± standard deviation from three independent experiments (Unpaired 2-tailed Student’s t-test, p = 0.04, t=2.445, df=8 for EZH2; p=0.014, t=3.095, df=8 for AGO1). I) RIP using DDK antibody (Origene) followed by qPCR detection of the Egr2-AS-RNA. Loss of Ser 118 inhibits the binding of YY1 to Egr2-AS-RNA as compared to non-mutated YY1-DDK. However, loss of Ser 184 induced a significant increase in binding of YY1 to the Egr2-AS-RNA, while Ser 247 has no effect compared to the non-mutated construct. The double mutation of Ser 118 and 184 results in increased binding of YY1 to the Egr2-AS-RNA compared to the non-mutated protein. The experiment was repeated three times and the results are presented as average ± standard deviation (One-Way ANOVA (5,12) = 3.398, p = 0.0383) followed by post-hoc Dunnett test (*p = 0.0281). J) Schematic representation of the AS-RNA-mediated regulation of Egr2 expression. In non-injured nerves NRG1 signaling to ERK1/2 phosphorylates YY1, which in turn activates the transcription of Egr2 and represses the expression of the AS-RNA (left panel). Nerve injury inhibits the NRG1-ERK1/2 signaling axis, which in possible conjunction with protein phosphatases blocks the Ser184 phosphorylation of YY1. This results in activation of the YY1 phospho-switch and association of non-phosphorylated YY1 with the AS-RNA and activation of AS-RNA expression. Loss of YY1 phosphorylation and increase in the expression of the AS-RNA results in inhibition of Egr2 transcription (right panel).

NRG1 induced ERK1/2 signaling leads to YY1-mediated regulation of the Egr2-AS-RNA

Recently, it was shown that the transcription factor YY1 is part of the ERK1/2 signaling pathway responsible for the upregulation of EGR2 in response to NRG1 in SCs (He et al., 2010). We examined if the increase in Egr2-AS-RNA expression after inhibition of ERK1/2 signaling (Figure 6C) depends on YY1 modulation of the Egr2-AS-RNA. We identified a 100 nucleotide-long region of the Egr2 promoter upstream of the transcription start site flanking the 5′- of the Egr2-AS-RNA (Figure 6D) that contains a previously described YY1 binding motif on the antisense strand (He et al., 2010) and could possibly regulate the expression of the Egr2-AS-RNA (S1 region, Figure S5A). To determine whether YY1 directly associates with this region in living cells, we performed chromatin immunoprecipitation (ChIP). We then tested the recruitment of YY1 to the S1 region of the Egr2 promoter and to a separate region (S2) located further upstream on the Egr2 promoter (between nucleotides −723 and −647 relating to the transcription start site of Egr2) that contains a conserved YY1 binding motif. YY1 was recruited only to the S2 region of the Egr2 promoter in SCs incubated in the presence of NRG1, which agrees with previously reported data on YY1 binding on Egr2 promoter (He et al., 2010). Inhibition of ERK1/2 signaling with UO126 results in partial but significant inhibition of YY1 interaction with the Egr2 promoter (Figure 6E). Next, we examined if YY1 associates with the S1 and S2 regions of single stranded RNA using RNA immunoprecipitation (RIP). Inhibition of ERK1/2 signaling with UO126 results in significantly increased association of YY1 with S1 and S2 regions of the Egr2-AS-RNA (Figure 6F). Our data indicate that NRG1-ERK1/2 signaling increases YY1 binding to the Egr2 promoter and activates Egr2 transcription while represses expression of the Egr2-AS-RNA. Inhibition of ERK1/2 signaling results in de-repression of the Egr2-AS-RNA expression (Figure 6C) through increased binding of YY1 to the S1 and S2 regions of the Egr2-AS-RNA (Figure 6F) and inhibition of Egr2 expression (Figure 6C).

YY1 regulates binding of the Egr2-AS-RNA to EZH2

Since inhibition of ERK1/2 signaling in SCs increases binding of YY1 to the Egr2-AS-RNA (Figure 6F) we tested if YY1 affects the functional interactions of the Egr2-AS-RNA with chromatin remodeling complexes. It has been previously determined that EED and EZH2 are core components of a multi-subunit histone methyltransferase complex, PRC2, with specificity for lysine 27 (H3K27) of histone H3 (Cao et al., 2002; Czermin et al., 2002; Muller et al., 2002). We hypothesized that the Egr2-AS-RNA interacts physically with protein components of the PRC2 and that YY1 mediates this interaction. We silenced total YY1 expression in SCs using siRNAs targeting four separate areas of the YY1 sequence (Figure 6G). Then we performed RIP with ChIP-validated antibodies against EZH2, AGO1, AGO2 and H3K27me3 using RNA from YY1 siRNA or non-targeting siRNA transfected SCs. In control SCs (non-targeting siRNA transfected) the Egr2-AS-RNA binds and precipitates exclusively with EZH2 (Figure 6H). Following YY1 knockdown in YY1 siRNA transfected SCs we detected complete loss of binding of the Egr2-AS-RNA to EZH2 and increased binding of the Egr2-AS-RNA to AGO1 (Figure 6H). This may affect turnover of the Egr2-AS-RNA since AGO proteins have been previously implicated in non-coding RNA turnover (Yoon et al., 2015), or indicate a “switch” to the Egr2-AS-RNA function from RPC2-mediated chromatin remodeling to AGO-mediated transcriptional silencing (Janowski et al., 2006). Finally, we show that inhibition of YY1 inhibits Egr2 mRNA expression and induces the expression of the Egr2-AS-RNA (Supplementary Figure S5B).

Phosphorylation state of YY1 regulates binding to the Egr2-AS-RNA

Recently it was shown that NRG1 mediated MEK-ERK1/2 signaling induces phosphorylation of YY1 at Ser 118, 184 and 247 and this phosphorylation has a key role in regulating Egr2 transcription (He et al., 2010). We hypothesized that binding of YY1 to the Egr2-AS-RNA may be regulated by the state of phosphorylation of YY1 Serine residues. We generated several Ser-Ala mutations at positions 118, 184, 247 and a double mutation at positions 118 and 184. To determine the effect of the loss of each Serine on the binding of YY1 to Egr2-AS-RNA we performed RIP followed by qPCR detection of the Egr2-AS-RNA. We show that loss of Ser 118 results in significant inhibition of the binding of YY1 to Egr2-AS-RNA as compared to non-mutated YY1. However, loss of Ser 184 induces a significant increase in binding of YY1 to the Egr2-AS-RNA, while Ser 247 has no effect compared to the non-mutated construct (Figure 6I). Finally, the double mutation of Ser 118 and 184 results in increased binding of YY1 to the Egr2-AS-RNA compared to the non-mutated protein suggesting that Ser 184 is the dominant regulatory site (Figure 6I).

Discussion

Studies on genetically modified mice (Decker et al., 2006; Le et al., 2005; Topilko et al., 1994) and identification of the mutations associated with peripheral neuropathies (Bellone et al., 1999; Timmerman et al., 1999; Warner et al., 1998) have implicated EGR2 as a central regulator of peripheral myelination (Svaren and Meijer, 2008). During myelination, various SC genes are dynamically regulated and the majority of these genes are targets of EGR2 transcriptional control (D’Antonio et al., 2006; Jang et al., 2010; Nagarajan et al., 2001; Verheijen et al., 2003). Recently the transcription factor YY1 has been implicated as a molecular link between extracellular signals and the regulation of EGR2 expression (He et al., 2010). Although the importance of trans-acting proteins (e.g. transcription factors) has been established, the existence of an epigenetic circuit that allows SCs to regulate the expression of EGR2 in response to injury and during myelination has not been previously described. The discovery that the majority of eukaryotic genomes are transcribed (Consortium et al., 2007) and that many of the resulting transcripts are developmentally regulated (Mercer et al., 2008) but do not encode proteins (Simon et al., 2011) has steered our attention toward the role of long non coding RNA in the regulation of Egr2 transcription.

We discovered a cis-acting long noncoding RNA antisense to the promoter of Egr2. Since ectopic expression of the Egr2-AS-RNA inhibits the expression of Egr2 mRNA we asked if the Egr2-AS-RNA exerts reversible regulation of Egr2 expression during peripheral nerve injury. During the acute phase of nerve injury response, the expression of EGR2 is inhibited and demyelination ensues (Guertin et al., 2005; Parkinson et al., 2008). We determined that the Egr2-AS-RNA mediates the inhibition of Egr2 mRNA expression, while inhibition of the Egr2-AS-RNA expression results in delayed demyelination even after complete nerve transection. In addition, inhibition of the Egr2-AS-RNA expression restores the Egr2 transcript expression levels and rescues the EGR2-regulated gene expression profile in the injured nerves. These data raised a series of questions regarding how the expression of the Egr2-AS-RNA is regulated and how the Egr2-AS-RNA is integrated within the pathways that control nerve injury response. It has been shown that c-Jun is an essential transcription factor for the reprogramming of mature myelinating SCs to de-differentiated SCs after nerve injury (Arthur-Farraj et al., 2012). c-Jun inhibits the Egr2 mediated myelin gene expression (Parkinson et al., 2008) and is a negative regulator of myelination, which suggests a possible interplay between the Egr2-AS-RNA expression and c-Jun during the acute nerve injury response.

The expression of EGR2 depends on NRG1 mediated ERK1/2 signaling to YY1 during peripheral myelination (He et al., 2010; Newbern et al., 2011). We identified a portion of the Egr2 promoter adjacent to the transcription start site (TSS) that fulfills the criteria for a bidirectional promoter containing a TATA box and a YY1 binding motif (Smale and Kadonaga, 2003). Although, this bidirectional promoter affects expression of the Egr2-AS-RNA we cannot target it to generate mice lacking expression of the Egr2-AS-RNA since this approach will also affect expression of the Egr2 mRNA through elimination of the TATA box and several TF binding sites (Rangnekar et al., 1990). YY1 has been previously implicated in the transcriptional activation of Xist during the initiation and maintenance of X-inactivation through direct activation of the Xist promoter (Makhlouf et al., 2014). It has also been shown that YY1 is an RNA binding protein and binds Xist as an adaptor protein between the lncRNA and chromatin targets (Jeon and Lee, 2011). Here we discovered that YY1 mediates the binding of the Egr2-AS-RNA to EZH2, which is the core component of the PRC2 chromatin remodeling complex. How this function of YY1 is regulated and what is the biologic significance during nerve injury response is unknown. It could be possible that YY1 functions as a molecular scaffold that coordinates targeting of the Egr2-AS-RNA to PRC2 and chromatin thereby coupling expression of the Egr2-AS-RNA with transcriptional repression.

The various functions of YY1 can be modulated by post-translational modifications including phosphorylation (Rizkallah and Hurt, 2009). We describe here, a YY1 phospho-switch mechanism (schematic at Figure 6J) that regulates binding of YY1 to Egr2 mRNA or the Erg2-AS-RNA. We identified Ser184 as the regulatory site to induce binding of YY1 to the Egr2-AS-RNA. It has been shown that AURORA B kinase phosphorylates Ser184 of YY1 during G2/M transition of the cell cycle and that Protein Phosphatase 1 (PP1) rapidly dephosphorylates YY1 at Ser184 (Kassardjian et al., 2012). It is possible that dephosphorylation of YY1 following peripheral nerve injury is cell cycle dependent as SCs dedifferentiate and that PP1 plays a role in this process.

The speed at which a cell responds to an extracellular cue by activating a set of genes and repressing another is of pivotal importance to the fate of that cell. However, this aspect of gene regulation is often not appreciated. Instead, the absolute levels of expression are generally seen as the hallmarks of a regulated gene (Uhler et al., 2007). Here we have shown that an antisense RNA transcript that associates with Egr2 promoter in cis regulates the levels of Egr2 transcription in response to extracellular signals. We propose that the Egr2-AS-RNA confers transcriptional buffering to maintain the proper levels of Egr2 transcription. Given that non-coding AS-RNAs are often expressed in a tissue- or time-dependent manner, the mechanism of Egr2-AS-RNA regulation of Egr2 transcription involves chromatin remodeling and affects the rate of Egr2 induction rather than the steady-state levels of gene expression. In fact, we have shown that the Egr2-AS-RNA gradually recruits a chromatin remodeling complex on the Egr2 promoter and its role in chromatin plasticity and transcriptional silencing of Egr2 is instructive since inhibition of the Egr2-AS-RNA results in the dissociation of the remodeling complex from the Egr2 promoter.

We have identified an antisense RNA that is induced after nerve injury and regulates the transcription of Egr2 as part of an NRG1-ERK1/2-YY1 signaling axis. This functional exploration of an antisense long non-coding RNA in Schwann cell biology will likely have a major impact on our understanding of the transcriptional regulation of peripheral myelination.

Experimental Procedures

Rapid amplification of cDNA ends (5′-RACE)

For 5′-RACE, we used the RLM RACE kit from Ambion with certain modifications. Total RNA was treated with Calf Intestine Alkaline Phosphatase (CIP) to remove free 5′-phosphates from molecules such as ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA. The cap structure found on intact 5′ ends of mRNA is not affected by CIP. The RNA was then treated with Tobacco Acid Pyro- phosphatase (TAP) to remove the cap structure from full-length mRNA, leaving a 5′-monophosphate. A 45 base RNA Adapter oligonucleotide provided by Ambion was ligated to the RNA population using T4 RNA ligase. The adapter cannot ligate to dephosphorylated RNA because these molecules lack the 5′-phosphate necessary for ligation. During the ligation reaction, the majority of the full length, decapped mRNA acquires the adapter sequence as its 5′ end. We then used random sense decamers that will bind to the antisense strand and a primer antisense to the 5′-adapter in order to amplify the AS-RNA.

Computational verification of the Egr2-AS-RNA expression

RNA-seq raw reads from mouse sciatic nerves in samples SRR3222412, SRR3222413 and SRR3222414 were downloaded from the GEO Data Sets: (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM2086001). Reads were aligned to the mm10 assembly of the mouse genome with gsnap (Wu and Nacu, 2010). Read mapping to genes was done based on the refSeq exons, as defined in file http://hgdownload.cse.ucsc.edu/goldenPath/mm10/database/refGene.txt.gz. The coordinates of the AS-RNA on chr10, were added to the exon file. Read summarization was done with featureCounts (Liao et al., 2014) and raw read counts were standardized to the Reads-per-Kilobase-per Million measure (Mortazavi et al., 2008) using reads with a mapping quality of 20 or better. To estimate the coordinates of the mature RNA, we first looked at the sequenced reads around the appropriate genomic location using the mpileup routine of SAMtools (Li et al., 2009) and then we visualized the reads using the Integrative Genomics Viewer (Robinson et al., 2011) as confirmation.

Chromatin isolation from sciatic nerves

2 month old mice were subjected to sciatic nerve transection. 48 hr after injury, the animals were euthanized and both nerves (distal part of the lesioned nerve and a 0.5 mm fraction of the contralateral healthy nerve) were isolated. Nerves (5 per condition) were then crosslinked with 1% PFA in PBS and neutralized with glycine (0.3M final concentration). Nerves were centrifuged at 2000 rcf for 5 min and washed twice with cold PBS plus protease and nuclease inhibitors. IP Lysis Buffer was added to the nerves (EpiTecht® ChIP OneDay Kit, QIAGEN, Venlo, Netherlands) and the nerves were homogenized mechanically for 15 sec with a Pro 200 homogenizer (Proscientific Inc.). Next, the samples were sonicated on ice using Misonix Sonicator 3000 (Fisher Scientifc), for 9 cycles of 30 sec ON and 90 sec OFF at 80% power to shear the chromatin between 100–1000bp. For ChIPs using SCs, we used components of the EpiTecht® ChIP OneDay Kit. Chromatin was sonicated to an average length of 1–2 kb on ice and centrifuged. The supernatant was used either for RNA and DNA ChIP.

DNA ChIP

Lysates were incubated O/N at 4°C on rotation with the following ChIP verified antibodies: Ezh2, Ago1 and Ago2 (Cell Signalling, Danvers, MA, USA) at a 1:50 dilution; H3K27 (Millipore, Billerica, MA, USA) at a dilution of 1:25, and without antibody as a control. Chromatin was then precipitated and DNA was extracted (EpiTecht® ChIP OneDay Kit, QIAGEN, Venlo, Netherlands). Recovered material from the input sample and all the ChIP samples per condition were used to perform qPCR of the Egr2 AS-RNA (primer sequences at Supplemental Information). Relative enrichment for each experimental sample was calculated as a percentage of the input. For negative control ChIPs we used a non-targeting isotype matched IgG and the values in all experiments ranged between 0–0.002% of the input sample. These values were used to normalize the data obtained with the target-specific antibodies. For all qPCRs reported in the paper, we performed a no-RT control amplification to verify the absence of genomic DNA contamination.

RIP

To perform the RIPs, we used the Magnetic Chromatin Immunoprecipitation Kit (RNA ChIP-IT®, Active Motif, Carlsbad, CA, USA). The antibodies used, analysis and plotting were performed as described for DNA ChIP.

Statistical Analysis

To determine statistical significance between the means of three or more independent groups we used one-way Anova. The homogeneity of variances was confirmed with Brown and Forsythe test and the significance between specific groups was calculated with a post-hoc Dunnett test. This analysis was performed for the data in figures: Fig 1C & 1D, Fig 3A & 3B, Fig 4C, Fig 6I. For the rest of the data we used unpaired two-tailed t-test. To verify Gaussian distribution of the data before applying the t-test, we performed the D’Agostino & Pearson and the Shapiro-Wilk normality tests. Statistical analysis was performed using Graphpad Prism.

Animal Use and Care

8-week-old male and female C57/B6 WT mice (gender does not affect peripheral nerve injury response) were obtained from Jackson Labs and maintained according to the NIH Guide for the Care and Use of Laboratory Animals. All animal use protocols were approved by the Institutional Animal Care and Use Committee of the Weis Center for Research, Geisinger Clinic.

Western Blots

The full scans of all western blots presented in the paper are included in Supplementary Figure S6.

Methods for Sciatic nerve injury, Lentivirus production, Mouse DRG explant and purified Schwann cell cultures, Immunocytochemistry protocols, RT-PCR and qPCR, Western blotting, Preparation of the hydrogel, ISH, Nuclear Run-on assay, PCR array, Electron Microscopy, siRNA transfections and Mutagenesis are included in detail in Supplemental Information.

Supplementary Material

supplement

Highlights.

  • Expression of Egr2 in peripheral nerves is regulated by a long non-coding RNA.

  • Egr2-AS-RNA gradually recruits an epigenetic silencing complex on the Egr2 promoter.

  • The Egr2-AS-RNA regulates nascent transcription of Egr2.

  • Expression of the Egr2-AS-RNA is regulated by Erk1/2 signaling to YY1.

Acknowledgments

We thank Dr. Steven Toms and members of the Tapinos lab for critically reviewing the manuscript. We also thank Dr. Fajardo for computational analysis of the RNAseq data. This work was supported from internal funds of the Geisinger Clinic to N.T. The authors declare no conflict of interest.

Footnotes

Author Contributions

Conceptualization, N.T.; Investigation, M.M.M., T.M.O., J.P.Z., A.O., and J.K.N.; Writing-Original Draft, N.T., and M.M.M.; Writing-Review and Editing, N.T.; Funding Acquisition, N.T.; Resources, N.T., and R.L.; Supervision, N.T., and R.L.

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