JBC Papers in Press. Published on January 22, 2015 as Manuscript M114.622878 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.622878 ChIP-seq Analysis of Peripheral Nerve Injury

Dynamic Regulation of Schwann Cell Enhancers after Peripheral Nerve Injury Holly A. Hung1, 2, Guannan Sun3, Sunduz Keles3, John Svaren1, 4 From 1Waisman Center, 2Cellular and Molecular Pathology Graduate Program, 3Department of Biostatistics & Medical Informatics, 4Department of Comparative Biosciences. University of WisconsinMadison, Madison, WI 53705, USA Running Title: ChIP-seq Analysis of Peripheral Nerve Injury To whom correspondence should be addressed: John Svaren, 1500 Highland Ave., Waisman Center, University of Wisconsin-Madison, Madison, WI 53705 Tel: 608 263 4246 Email:[email protected] Keywords: ChIP-sequencing (ChIP-seq), Schwann cells, histone modification, gene regulation, neurobiology, glial cell

ABSTRACT Myelination of the peripheral nervous system is required for axonal function and long term stability. After peripheral nerve injury, Schwann cells transition from axon myelination to a demyelinated state that supports neuronal survival and ultimately remyelination of axons. Reprogramming of gene expression patterns during development and injury responses is shaped by the actions of distal regulatory elements that integrate the actions of multiple transcription factors. We used ChIP-seq to measure changes in histone H3K27 acetylation, a mark of active enhancers, to identify enhancers in myelinating rat peripheral nerve, and their dynamics after demyelinating nerve injury. Analysis of injury-induced enhancers identified enriched motifs for c-Jun, a transcription factor required for Schwann cells to support nerve regeneration. We identify a cJun-bound enhancer in the gene for Runx2, a transcription factor induced after nerve injury,

Myelination of the peripheral nervous system is a vital aspect of nervous system development and function. The myelin sheath produced by Schwann cells is a specialized multilayer membrane essential for the rapid transmission of nerve impulses by axons. Impaired myelin structure and function is a causative factor in a number of peripheral nerve disorders, such as Charcot-Marie-Tooth Disease (CMT). Schwann cells not only produce and maintain the myelin sheath but also retain the developmental plasticity to break down myelin in response to injury, secrete molecules to support axonal survival, and then eventually remyelinate axons in regenerating nerves (1-3).

1 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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and show that Runx2 is required for activation of other induced genes. In contrast, enhancers that lose H3K27ac after nerve injury are enriched for binding sites of the Sox10 and Early Growth Response 2 (Egr2/Krox20) transcription factors, which are critical determinants of Schwann cell differentiation. Egr2 expression is lost after nerve injury, and many Egr2 binding sites lose H3K27ac after nerve injury. However, the majority of Egr2bound enhancers retain H3K27ac, indicating that other transcription factors maintain active enhancer status after nerve injury. The global epigenomic changes in H3K27ac deposition pinpoint dynamic changes in enhancers that mediate the effects of transcription factors that control Schwann cell myelination and peripheral nervous system responses to nerve injury.

Background: Nerve injury induces Schwann cells to remove myelin sheaths, and secrete factors that stimulate nerve regeneration. Results: ChIP-seq was used to identify injuryregulated enhancers in peripheral nerve. Conclusion: Dynamically-regulated enhancers identify target sequences of injury-regulated transcription factors in Schwann cells. Significance: These data elucidate transcription factor pathways that coordinate Schwann cell responses to peripheral nerve injury.

ChIP-seq Analysis of Peripheral Nerve Injury

EXPERIMENTAL PROCEDURES Nerve injury surgery. All animal experiments were performed according to protocols approved by the University of Wisconsin, School of Veterinary Medicine (Madison, WI). Male P25 Sprague Dawley rats or adult male mice (Harlan Laboratory) were housed with a normal light/dark cycles and ad libitum access to food and water. Prior to surgery, animals were anesthetized with isoflurane (Piramal Healthcare), and an injection of 5mg/kg of ketoprofen was given for analgesia. Under aseptic conditions, an incision (~5mm long) was made through the skin and sciatic nerve at the proximal lateral region of the femur. As a control, the contralateral leg also received a sham operation consisting of only a skin incision. The

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acetylases CBP/p300 (27). Recently, the chromatin mark for acetylated histone H3 lysine 27 (H3K27ac) has been identified as an epigenetic marker of actively engaged enhancer elements (28-30), and allows differentiation from other nucleosome-depleted regions of chromatin, such as poised enhancers. Substantial progress has been made in profiling gene expression changes and establishing the regulatory hierarchy and target genes of individual transcription factors during development and injury of peripheral nerve (9,23,31-33). Previous studies have elucidated combinatorial control of individual enhancers driving Schwann cell differentiation genes, such as Gjb1, Mbp, Mpz Egr2, and Sox10 (4,12,14,15,3440). However, genome-wide identification of the enhancers that form the substrate of transcription factors’ effects on gene expression is required to understand targeting mechanisms, and to reveal the combinatorial control of genome-wide regulatory networks underlying myelin formation and the Schwann cell’s response to nerve injury. To identify active regulatory elements in Schwann cells, the chromatin signature H3K27ac was assessed in mature peripheral nerve tissue and after injury-induced demyelination. These data identified injury-regulated enhancers that mediate the effects of transcription factors that decline or increase after nerve injury. Furthermore, transcriptional regulators of Schwann cell differentiation were identified through analysis of enhancers targeted for epigenomic reprogramming that occurs after a demyelinating injury.

Induction and maintenance of Schwann cell myelination is mediated by several key transcription factors (4,5). For example, Early Growth response 2 (also known as Krox20) is a C2H2 zinc-finger transcription factor required for myelin formation and maintenance by Schwann cells (6-8) and has been shown to regulate several major myelin genes (8,9). Sox10 is a critical HMG box transcription factor for Schwann cell specification and development, and is also required for maintenance of peripheral nerve myelination (10). Egr2 and Sox10 activate myelin gene transcription through coordinate binding of distal and intronic enhancer sites in critical myelin genes such as Connexin 32 (Gap Junction B1/Gjb1), Myelin Basic Protein (Mbp), Peripheral Myelin Protein 22 (Pmp22) and Myelin Protein Zero (Mpz) (11-16). The breakdown of axon-Schwann cell contact following nerve injury results in reduced expression of major myelin genes, which can be attributed in part to a rapid loss of Egr2 expression (17,18). Several studies have highlighted the involvement of specific signaling pathways in mediating demyelination after nerve injury, such as p38, JNK and ERK signaling (19-22). JNK and p38 signaling activate c-Jun, a basic leucine zipper transcription factor that is one the major transcriptional determinants that trigger rapid demyelination in Schwann cells and induce a genetic program that supports axon regeneration (23). Gain of c-Jun and loss of Egr2 are therefore major contributors to altered gene expression in Schwann cells of injured nerve. Gene expression changes during development and injury are shaped by the actions of enhancers that integrate the actions of multiple transcription factor inputs to regulate associated genes. These distal enhancer elements are integration hubs for signaling pathways activated by extracellular signals, and sequence analysis of active enhancers can be used to determine binding motifs for transcription factors that regulate enhancer activity (24). Recent genomics studies using chromatin immunoprecipitation (ChIP-seq) have offered tools to assess not only transcription factor binding but also enhancer activation status in specific tissues in a stage-dependent manner (25,26). General characteristics of enhancers include open chromatin, histone H3K4 monomethylation, and recruitment of histone

ChIP-seq Analysis of Peripheral Nerve Injury blocking procedure. ChIP DNA was purified by phenol chloroform extraction and resuspended in 10mM Tris pH 8.0. Antibodies used in the study: H3K27ac (Active Motif, 37133), normal rabbit IgG (Millipore, 12-370), cJun H-79 (Santa Cruz, SC-1694). Statistics were calculated using Student’s t-test. Error bars represent standard deviation and asterisks denote p-value (* p≤ 0.05, ** p≤ 0.005). The samples were generated from independent chromatin pools (n = 3 for transcription factor ChIP, n = 2 or 3 for H3K27ac ChIP) and were analyzed using quantitative PCR listed in Table 1.

skin wound was sutured with rodent surgical staples, and the rats were caged for 72hr post operation. The nerve tissue distal to the transection or sham site was isolated for use in expression and ChIP experiments.

ChIP-seq. Libraries were prepared from sham/injured P25 sciatic nerve H3K27ac ChIP samples, and respective inputs, using the Illumina TruSeq ChIP Sample Preparation Kit (Illumina Inc., San Diego USA). After library construction and amplification, quality and quantity were assessed using an Agilent DNA 1000 series chip assay (Agilent Technologies, Santa Clara, CA, USA). The libraries were sequenced using Illumina HiSeq2000. Basecalling was performed using the standard Illumina Pipeline. Reads were mapped to the Rattus norvegicus genome Rn5 using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) to produce SAM files for further analysis. From the two biological replicates, we obtained 33,244,433 and 24,935,382 (Sham) and 21,485,993 and 45,718,907 (Injury) uniquely mapping reads. Input runs mapped 39,351,486 and 21,297,471 (Sham) and 42,439,666 and 38,164,305 (Injury) reads.

Chromatin immunoprecipitation (ChIP). Freshly dissected rat sciatic nerve was minced in 1% formaldehyde for 10min and then quenched for 10min with glycine to a final concentration of 0.125M. Samples were sequentially lysed in buffers LB1, LB2, and LB3 + 0.03% SDS (41). DNA was fragmented to an average size of 0.52kb using 4x10min Bioruptor (Diagenode) cycles on the medium setting. Each aliquot of sonicated chromatin (300ug) was incubated overnight at 4°C with 5ug of antibody. A 10% aliquot was saved for input analysis. For histone antibody ChIP experiments, an 80ul aliquot of Protein G Dynabead (Invitrogen) slurry was added to each ChIP sample for 4hrs at 4°C. IPs were washed 5x in RIPA buffer then eluted at 65°C in reverse crosslinking buffer (50mM Tris, 10mM EDTA, 1% SDS). For transcription factor antibody ChIP experiments, chromatin-antibody mixtures were incubated in Protein G sepharose beads (GE healthcare) were used as previously described (12,42), excluding herring sperm DNA from the

Peak Calling. MOSAiCS (43) was used to determine enriched binding regions for Sham_H3K27ac Injury_H3K27ac ChIP, respectively. For all data sets, BED files were generated with HOMER and subsequently uploaded to the UCSC genome browser for visualization purposes. Peak finding performed using the R package MOSAiCS (MOdel-based one and two Sample Analysis and inference for ChIP-seq Data) controlled for a false discovery rate (FDR) at 0.05. MOSAiCS implements a model-based approach where the background distribution for unbound regions takes into account systematic biases such as mappability and GC content and the peak regions are described with a two component Negative Binomial mixture model.

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Immunohistochemistry (IHC) Freshly dissected mouse sciatic nerve from sham and distal sections of injured nerve were fixed with 4% paraformaldehyde for 4hrs at 4C then immersed in PBS overnight at 4C. The nerve samples were embedded in Tissue-Tek OCT compound (Sakura Finetek) and cut into 10 μm cryostat sections. For fluorescence immunohistochemistry, the sections were blocked in 3% donkey serum/0.2% Triton X-100 for 1 h at room temperature. Incubation with primary antibody was performed overnight at 4°C in blocking solution, and secondary antibody incubation was performed at room temperature for 1 h. The primary antibodies used were rabbit antiRunx2 (1:100; Santa Cruz SC10758), goat antiSox10 (1:200; R&D Systems AF2864). Secondary antibodies used were anti-rabbit Alexa 488 and anti-goat Alexa 568 (1:1000; Invitrogen, A21206 and A11057). Confocal images taken using Leica TCS LSI Macro microscope (Leica, Wetzlar, Germany).

ChIP-seq Analysis of Peripheral Nerve Injury in 1:1 Serum-free DMEM: Hams F12, with insulin/transferrin/selenium supplement (Sigma). RNA was purified from cells 48hr after transfection using Tri Reagent (Ambion) and analyzed by quantitative reverse transcription (RT)-PCR using the StepOne Plus system (Applied Biosystems). Relative amounts of each gene were determined by the comparative CT method and normalized to 18S rRNA (47). Statistics were calculated using Student’s t-test. Error bars represent standard deviation and asterisks denote p-value (* p≤ 0.05, ** p≤ 0.005). The statistics were generated from independently generated samples in triplicate (n = 3) and were analyzed using primers listed in Table 2.

Statistically different peaks were determined using DBChIP (44). Peak files are deposited in NCBI GEO under accession number GSE63103. Functional enrichment and Genome Ontology Analysis. DAVID (Database for Annotation, Visualization, and Integrated Discovery) is an integrated biological knowledgebase and gene annotation tool set for the identification and clustering of statistically relevant functional and biological categories in large gene sets. Gene Ontology (GO) categories were clustered using high stringency and categories with p-values 2-fold after nerve injury had a nearby annotated InjuryDB peak. In contrast, InjuryDB peaks were associated with only ~13% of genes expressed more highly in the Sham condition (i.e. repressed after injury), whereas ShamDB peaks were associated with >30% of genes in this class (Figure 3C). A limitation of this analysis is that enhancers often do not regulate the nearest gene, but the prevalence of differential peaks is generally correlated with increased and decreased gene sets in nerve injury.

ChIP-seq Analysis of Peripheral Nerve Injury

Gene ontology analysis of Egr2 and Sox10 enhancer elements. To collectively assess the H3K27acmarked binding sites for Egr2 and Sox10, we annotated each distal enhancer peak to the nearest transcription start site and calculated the enrichment of gene ontology (GO) terms for functional groupings. Egr2 peaks colocalizing with H3K27ac were associated with genes involved in axon ensheathment and regulation of locomotion. H3K27ac-marked Sox10 peaks were enriched near genes involved in regulation of nerve impulse transmission and axon ensheathment (Figure 4). Interestingly, Kegg pathway analysis identified Focal Adhesion and Adherens Junction categories. Lipid-rich myelin contains not only major myelin protein but also a rich assortment of anchoring junction proteins. Anchoring junctions such as gap junctions and adherens junctions are traditionally found to link two cells, but in peripheral myelin, they are found on opposing membrane layers. All these junction ontologies were enriched in the Sox10 peak sets. Further exploration of focal adhesion genes that encode proteins linking Schwann cells to the extracellular matrix identified several laminins and their integrin receptors. Schwann cell myelination requires the integration of laminin activities for process extension and morphogenesis (58-63). Laminins 411 and 211 (encoded by Lama4, Lama2, Lamb1, and Lamc1 genes) interact with integrin receptors, such as Integrin α6 (Itga6), during the process of radial sorting (64,65). All of these genes have Sox10-bound enhancers (see Lamb1 and Itga6 in Figure 4C) and are regulated by Sox10 in siRNA studies of the S16 Schwann cell line (51). Another laminin receptor is dystroglycan (Dag1), which interacts with the actin cytoskeleton and is required for Cajal band formation (50,58,66). Dystroglycan also interacts with Periaxin and Drp2 (50), and all 3 of these genes have prominent Sox10 binding sites (Figure 4C), and are regulated by Sox10 (51). Nerve injury effects on Egr2-bound enhancers. Consistent with the loss of Egr2 expression after nerve injury (17,18), several Egr2

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binding sites lose H3K27ac after nerve injury. An example is shown for the Myelin-associated Glycoprotein (Mag) gene (Figure 5A), which was previously shown to have Egr2 and Sox10 binding within a conserved intronic region (39,40,42). These sites are flanked by H3K27ac, but the active histone mark is practically abolished at 3 days after nerve injury. Similar reductions in H3K27ac are observed in intronic regions of the Sema5a (Semaphorin 5a) and Fa2h (Fatty acid 2hydroxylase) genes, where Egr2 binding sites are associated with a series of ShamDB peaks. The Fa2h and Sema5a genes are induced in myelinating Schwann cells in an Egr2-dependent manner (8), and are downregulated after nerve injury (23). These data would suggest that Egr2 is required for H3K27 acetylation at these enhancers. Based on these examples, we had hypothesized that most Egr2-bound enhancers would lose H3K27 acetylation after nerve injury. However, we surprisingly found that only a minor percentage of Egr2 sites with H3K27 acetylation (~12%) lost acetylation after nerve injury (Table 3). Since it was possible that H3K27ac may be generally lost over all Egr2 sites, but only reach statistical significance at a minor subset of sites in the DBChIP analysis, we also calculated if reads in all Egr2 peak regions (summed over the genome) were reduced after nerve injury, but we did not find a statistical difference of H3K27ac reads in aggregated Egr2 peak windows. Overall, ~300 Egr2 peaks lost acetylation after nerve injury (Table 3), suggesting that other co-binding factors retain H3K27 acetylation at most Egr2 binding sites after nerve injury. The Egr2 peaks marked by H3K27ac were analyzed by motif finding, and we observed enrichment of several motifs besides Egr2 itself, including the TEAD and SOX motifs noted above (Figure 5C). This analysis also revealed a CREB-binding motif, which may reflect binding of CREB family members that could mediate GPR126-cAMP signaling that is required for myelination (67,68). Since Sox10 mRNA expression is generally unchanged after nerve injury (9,23), we anticipated that most of these sites would retain H3K27ac after nerve injury. Although this is true for most Sox10 sites, we did find that ~15% of Sox10/H3K27ac sites lost H3K27 acetylation after nerve injury (Table 3). InjuryDB enhancers bound by Egr2 and Sox10 are depicted in the heatmap

sites overlapping ShamDB locations for each factor are listed in Supplemental Tables S1 and S2.

ChIP-seq Analysis of Peripheral Nerve Injury

Analysis of injury-induced enhancers and the cJun pathway. Analysis of transcription factor motifs enriched in InjuryDB peaks revealed cognate sites for several factors upregulated in nerve injury (Figure 6A), such as c-Jun. Identification of the cJun/AP-1 binding site provides an additional validation for the data set, as c-Jun coordinates important aspects of the Schwann cell’s response to nerve injury (23,69,70). Microarray analysis showed 62 injury-induced genes were reduced >2-fold in c-Jun deficient mice after nerve injury (23). We found that 21 of these genes had InjuryDB peaks. Interestingly, c-Jun binding motifs were found in InjuryDB peaks proximal to several c-Jun target genes including Runx2 and Gdnf, which are induced over 40-fold after peripheral nerve injury (9,23). Glia cell-derived neurotropic factor (Gdnf) induction in Schwann cells plays a prominent role in supporting axons after nerve injury (70). The induction of the Runx2 transcription factor (also known as Core-binding factor a1) after nerve injury is consistent with enrichment of the RUNX-binding motif in InjuryDB peaks (Figure 6A). The RUNX-binding motif was found in injury-induced enhancers proximal to 13 of the cJun-dependent, injury-induced genes including Runx2 itself, Olig1, and Shh. While no role for Runx transcription factors has been shown in peripheral nervous system development or injury,

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Runx2 has been previously found to physically interact and cooperatively bind with c-Jun on both activator protein-1 (AP-1) and RUNX-binding sites (71). RUNX binding motifs were found in close proximity to c-Jun motif sites within the Runx2 gene. A histogram of c-Jun and Runx2 motif locations with respect to InjuryDB peak centers shows that the factor motifs are enriched at about 350bp from the centers of H3K27ac peaks (Figure 6B). While Runx2 has been shown to be induced in a c-Jun dependent manner (23), it has not been shown that Runx2 is induced in Schwann cells. Therefore, immunohistochemistry for Runx2 was performed in sham and 3d injured sciatic nerve (Figure 6C). Although there was some minimal staining in the Sham condition, we see a significant increase in Runx2 staining, which was colocalized with Schwann cell expression of Sox10, indicating that the Runx2 induction occurs in Schwann cells in peripheral nerve. To test the involvement of c-Jun and Runx2 in activation of injury-associated genes, we performed siRNA analysis of these two transcription factors in primary rat Schwann cells to determine effects on gene expression (Figure 6D,E). Previous experiments have shown that cJun is induced in primary Schwann cells cultured in serum free medium (72). Interestingly, c-Jun siRNA caused Schwann cells to adopt a morphology similar to that described for primary Schwann cells from c-Jun null mice (23), in which the normal bipolar morphology was changed to a more flattened cell type lacking processes (not shown). This was not observed with siRunx2. As anticipated from analysis of c-Jun in nerve injury (23), we found that reduction of c-Jun expression resulted in downregulation of several injury-induced genes: Sonic Hedgehog (Shh), Glial-derived neurotrophic factor (Gdnf), Olig1, Hmga1 and Runx2. Interestingly, downregulation of Runx2 had no effect on c-Jun expression, but did downregulate Shh and Olig1, suggesting that c-Jun and Runx2 coordinately activate at least some injury-induced genes. While c-Jun can alter cAMP--induced changes in differentiation markers (69,72), c-Jun and Runx2 siRNA did not change Egr2 and Mpz levels in the basal medium conditions used for this assay (not shown). To explore if the regulation of these genes are directly controlled by c-Jun we took a closer

plot representation of transcription factor bound DB sites (Figure 4B). One of the interesting aspects of this analysis is that only ~10% of the differentially bound sites that are lost after nerve injury were initially occupied by Egr2. This suggests that the nerve injury program in Schwann cells leads to displacement of other injurysensitive transcription factors. Using the motifs found in ShamDB peaks (Figure 3), the corresponding transcription factor genes were analyzed in nerve injury microarray studies to determine if their levels decline after nerve injury. There were ~2-fold reductions of Nur77 factors (Nr4a1 and Nr4a2), and in NF1A after nerve injury (23,32). However, there was no observed decrease in TEAD family transcription factors after nerve injury, although the binding and activity of these factors are commonly modulated by post-translational modifications.

ChIP-seq Analysis of Peripheral Nerve Injury

DISCUSSION Recent studies have highlighted pathways that underlie the developmental plasticity of Schwann cells to break down myelin in response to injury and then eventually remyelinate axons. Injury-induced gene expression changes direct a mature Schwann cell to dissolve its basement membrane, resume proliferation, support axonal regrowth, direct the regenerating axon across the site of injury, and finally remyelinate (2,3,76). Interestingly, while demyelination is initiated by axonal cues, the age-dependent decline in the efficiency of axon regeneration has recently been attributed to alterations in the Schwann cell gene expression network rather than age-dependent effects on the axonal response (77). Originally considered a dedifferentiation process, gene expression differences and functional analysis have shown that demyelinating

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Schwann cells are functionally distinct from immature Schwann cells (23). Since regulation of this process has important implications for the successful regeneration of axons, identification of key regulatory elements and the factors that bind them will help elucidate the connectivity of injuryinduced transcription factors with their cognate regulatory elements. A key regulator of the Schwann cell’s response to nerve injury is c-Jun, which drives demyelination and subsequent remyelination after nerve injury in Schwann cells. Recent work has identified c-Jun-dependent genes that are induced after nerve injury (23,69). Accordingly, our analysis uncovered a number of injury-induced enhancers in c-Jun dependent genes (such as Runx2, Figure 7D) that contain canonical binding sites for c-Jun/AP-1, and the cJun binding motif is enriched in injury-induced enhancers. While c-Jun plays a profound role in the Schwann cell’s response to nerve injury, many other transcription factors are induced. Some of them are c-Jun-dependent, such as Runx2, but others appear to be induced in a c-Jun-independent manner (23). Another pathway implicated in the Schwann cell’s response to nerve injury is the Erk pathway. For example, induced activation of the Raf-Erk pathway results in induction of demyelination, and many of the genes induced by Erk activation (Mcp1/Ccl2, Cxcl10, Timp1, TGFβ1, Megf10) appear to be distinct from c-jundependent genes (20,22). In particular, Ccl2 is a critical chemokine involved in recruiting macrophages to peripheral nerves after injury and in a peripheral neuropathy induced by a myelin gene mutation (78). Interestingly, the InjuryDB sites are enriched in binding sites for the ETS family of transcription factors, which are commonly activated by Erk kinases. In Schwann cells, Ets1 is induced after nerve injury (9,23) and Ets factors have been previously implicated in Schwann cell survival (79). Our data sets allow us to not only detect injury-induced enhancers but also to determine the fate of Egr2-bound enhancers after nerve injury (17,18,80). Previous studies have indicated that cJun antagonizes Egr2 expression (69,72). Egr2 is lost relatively soon after peripheral nerve injury (17,18), and its expression is regained as Schwann cells resume myelination. We had anticipated that Egr2-bound enhancers would be selectively lost

look at the enhancers over these gene loci. Figure 7A shows the location of induced enhancers in the Runx2 gene after nerve injury, which were validated using ChIP analysis of selected sites (Figure 7C). Several enhancers contain consensus binding sites for the c-Jun and Runx2 transcription factors (Figure 7B), and Runx2 binding has previously been observed in this locus (73). In addition, the ENCODE project has demonstrated binding of c-Jun at sites 1-3 at the homologous regions of the human RUNX2 locus by ChIP-seq (74). To test for c-Jun binding at InjuryDB sites containing cJun consensus binding motifs, we performed ChIP in sham and injured nerves. Using a c-Jun antibody used previously in the ENCODE project (75), we examined Runx2 InjuryDB peaks containing either c-Jun or Runx2 binding motifs and found detectable binding of c-Jun at two sites in the Runx2 gene, which was specific to the injury condition (Figure 7C). Interestingly, site 3 showed positive binding for cJun although only a Runx2 consensus motif was found at this site, which may be due to cooperative binding between Runx2 and c-Jun. In addition, we also identified binding sites for c-Jun within injury-induced enhancers proximal to the Shh, Hmga1, and Gdnf genes as well, consistent with regulation of these genes by c-Jun after nerve injury (23) (Figure 8). Overall, we found binding of cJun on 6 of the 8 sites examined in our analysis.

ChIP-seq Analysis of Peripheral Nerve Injury

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H3K27ac reveals those enhancers that specifically decommissioned or activated after nerve injury. While we speculate that enhancer changes are responsible for gene expression changes after injury, it remains quite possible that enhancers with constitutive H3K27ac may regulate gene expression by regulation of other steps of transcriptional regulation besides H3K27 acetylation. The H3K27 acetylation data were used to refine sites of Egr2 and Sox10 binding to those that bind to active enhancers. Approximately one third of Egr2 and Sox10 binding sites are colocalized with active enhancers. Binding sites without H3K27ac may be active earlier in embryonic Schwann cell development, although we find relatively little overlap with neural crest enhancers (24). We have investigated if these represent poised enhancers that could become active after nerve injury, but that is for the most part not the case (data not shown). Probably the most likely explanation is that Egr2 and Sox10 bind adventitiously to areas of open chromatin surrounding other types of elements in the genome (83). Our data also suggest that isolated binding of Egr2 or Sox10 is not sufficient to form an active enhancer region, since most clustered Egr2/Sox10 binding sites are positive for H3K27 acetylation. Moreover, most of the Egr2- and Sox10-bound enhancers surrounding major myelin genes are marked by H3K27ac, consistent with previous studies using H3K27ac as a mark of active enhancers (28,29). While previous ChIP-seq studies of peripheral nerve reflected binding of Schwann cell-specific factors (51), it is acknowledged that analysis of histone modifications in peripheral nerve is complicated by presence of non-Schwann cell types. Nonetheless, Schwann cells compose >80% of the nuclei in sciatic nerve (17,18), and colocalization of enhancer marks with Egr2 and Sox10 indicates that these enhancers are derived from Schwann cells. Overall, the differentiation state of Schwann cells is highly dependent on axonal signals (84), so analysis of intact nerve is best able to capture epigenomic consequences of axon-initiated signaling pathways. Histone acetylation is controlled by activity of both histone acetylases (principally CBP/p300 for H3K27ac) and deacetylases. Accordingly, ChIP-seq profiling of CBP/p300 has

after nerve injury. However, we surprisingly found that most Egr2-bound enhancers were retained, and only about 10% of the Egr2-bound enhancers lost the H3K27ac mark upon injury. This may be a minimum estimate since the cutoffs used to select differentially bound peaks are somewhat conservative. The maintenance of H3K27 acetylation on most Egr2-bound enhancers after injury could reflect compensatory binding of Egr2-related transcription factors that are induced after nerve injury, such as Egr1 (17). Alternatively, it is possible that the requisite histone acetylases are retained by other factors binding to the same enhancers, which may facilitate rebinding of Egr2 to these enhancers during remyelination. Our analysis highlighted other transcription factors whose binding motifs are enriched in injury sensitive enhancers. These include the NF1, Nur77 and TEAD binding motifs. As noted above, TEAD factors have been implicated in pathways that respond to neuregulin stimulation (55). Further studies will be required to elucidate the roles of these factors, since myelination depends on diverse transcription factors beyond Egr2 and Sox10 (81). It is anticipated that this enhancer analysis will help identify other transcription factors in Schwann cell development and their target enhancers. As noted above, we also found that some ShamDB enhancers were colocalized with Sox10 sites. The loss of acetylation at such sites could reflect coordinated binding of Sox10 with factors like Egr2 and Nfatc3/c4 (40,82), and potentially others that are lost after nerve injury. In addition, there may be competition with injury-induced Sox family members, such as Sox2 (3,8), which may repress a subset of Sox10-occupied enhancers. Using enhancer marks to identify regulatory regions has been used extensively in established cell lines and stem cells based on the ENCODE project and other efforts (24,25,28,29), but our studies have identified active regulatory elements in vivo using sciatic nerve as a ChIP-seq substrate. To our knowledge, no previous study has profiled dynamic enhancer changes during an injury process. The ChIP-seq analysis showed clear enhancer changes in major myelin genes that decrease upon nerve injury (Mag and other myelin genes), as well as injury-induced genes, such as Gdnf and Ngfr (70). As such, profiling of

ChIP-seq Analysis of Peripheral Nerve Injury

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noncoding regulatory elements constitute a significant percentage of the genome, and mutations and copy number variations affecting regulatory elements have been associated with a variety of genetic disorders (90-92). Only a few noncoding regulatory element mutations have been identified for hereditary peripheral neuropathies, including mutations of Sox10 binding sites in the Gjb1/Connexin 32 promoter (16,93) and recent identification of a mutation that is a candidate modifier of CMT1A (94). Until recently, technical limitations have limited mutational analysis to coding region mutations, but the exponential advances in sequencing technology have made it possible to identify tissue-specific regulatory elements that may harbor genetic lesions that cause or modify severity of peripheral nerve disorders.

also been used to identify regulatory elements genome-wide (85), and previous studies have shown an interaction between Egr2 and CBP/p300 (12,86). Histone deacetylase (HDAC) activity is required for Schwann cell differentiation (87,88). After nerve injury, it is possible that there is targeted deacetylation of specific enhancer regions, although the complexes and factors required for targeted deacetylation in nerve injury have not been identified. Identification of enhancers in peripheral nerve may also inform studies of the genetic basis of human peripheral neuropathy. While substantial progress has been made in identifying causes of genetic diseases such as CMT (89), most of the successes have resulted from a focus on coding regions. In contrast, analysis of the vast majority of noncoding DNA has been hampered by a lack of functional annotation for noncoding DNA. However,

ChIP-seq Analysis of Peripheral Nerve Injury

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Acknowledgements We thank Marie Adams at the UW Biotechnology Center for performance and preliminary analysis of Illumina sequencing, Xiao-yu Liu and the Bioinformatics Resource Center for bioinformatic assistance, and Karla Knobel for assistance in microscopy. We also thank Tony Antonellis for critical comments on the manuscript. FOOTNOTES *This work was supported by NIH grant NS075269 to J.S., and NIH HD03352 core grant.

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Taveggia, C., Feltri, M. L., and Wrabetz, L. (2010) Signals to promote myelin formation and repair. Nat Rev Neurol 6, 276-287 Visel, A., Blow, M. J., Li, Z., Zhang, T., Akiyama, J. A., Holt, A., Plajzer-Frick, I., Shoukry, M., Wright, C., Chen, F., Afzal, V., Ren, B., Rubin, E. M., and Pennacchio, L. A. (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854-858 Luciano, R. L., and Wilson, A. C. (2003) HCF-1 functions as a coactivator for the zinc finger protein Krox20. J Biol Chem 278, 51116-51124 Jacob, C., Christen, C. N., Pereira, J. A., Somandin, C., Baggiolini, A., Lötscher, P., Ozçelik, M., Tricaud, N., Meijer, D., Yamaguchi, T., Matthias, P., and Suter, U. (2011) HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells. Nat Neurosci 14, 429-436 Chen, Y., Wang, H., Yoon, S. O., Xu, X., Hottiger, M. O., Svaren, J., Nave, K. A., Kim, H. A., Olson, E. N., and Lu, Q. R. (2011) HDAC-mediated deacetylation of NF-κB is critical for Schwann cell myelination. Nat Neurosci 14, 437-441 Timmerman, V., Strickland, A. V., and Züchner, S. (2014) Genetics of Charcot-Marie-Tooth (CMT) Disease within the Frame of the Human Genome Project Success. Genes (Basel) 5, 13-32 Schaub, M. A., Boyle, A. P., Kundaje, A., Batzoglou, S., and Snyder, M. (2012) Linking disease associations with regulatory information in the human genome. Genome Res 22, 1748-1759 Maurano, M. T., Humbert, R., Rynes, E., Thurman, R. E., Haugen, E., Wang, H., Reynolds, A. P., Sandstrom, R., Qu, H., Brody, J., Shafer, A., Neri, F., Lee, K., Kutyavin, T., Stehling-Sun, S., Johnson, A. K., Canfield, T. K., Giste, E., Diegel, M., Bates, D., Hansen, R. S., Neph, S., Sabo, P. J., Heimfeld, S., Raubitschek, A., Ziegler, S., Cotsapas, C., Sotoodehnia, N., Glass, I., Sunyaev, S. R., Kaul, R., and Stamatoyannopoulos, J. A. (2012) Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190-1195 Kellis, M., Wold, B., Snyder, M. P., Bernstein, B. E., Kundaje, A., Marinov, G. K., Ward, L. D., Birney, E., Crawford, G. E., Dekker, J., Dunham, I., Elnitski, L. L., Farnham, P. J., Feingold, E. A., Gerstein, M., Giddings, M. C., Gilbert, D. M., Gingeras, T. R., Green, E. D., Guigo, R., Hubbard, T., Kent, J., Lieb, J. D., Myers, R. M., Pazin, M. J., Ren, B., Stamatoyannopoulos, J. A., Weng, Z., White, K. P., and Hardison, R. C. (2014) Defining functional DNA elements in the human genome. Proc Natl Acad Sci U S A 111, 6131-6138 Houlden, H., Girard, M., Cockerell, C., Ingram, D., Wood, N. W., Goossens, M., Walker, R. W., and Reilly, M. M. (2004) Connexin 32 promoter P2 mutations: a mechanism of peripheral nerve dysfunction. Ann Neurol 56, 730-734 Brewer, M. H., Ma, K. H., Beecham, G. W., Gopinath, C., Baas, F., Choi, B. O., Reilly, M. M., Shy, M. E., Züchner, S., Svaren, J., Antonellis, A., and (INC), t. I. N. C. (2014) Haplotypespecific modulation of a SOX10/CREB response element at the Charcot-Marie-Tooth disease type 4C locus SH3TC2. Hum Mol Genet 23, 5171-5187

ChIP-seq Analysis of Peripheral Nerve Injury

FIGURE LEGENDS Figure 1. Peak calling and analysis of H3K27ac marked active enhancers in myelinating nerve. A, ChIP-seq mapping of H3K27ac-containing enhancers was performed in vivo using distal nerve tissue from rat sciatic nerve 72hr post nerve transection or sham surgery. B, The Venn diagram illustrates the overlap between called peaks identified in Sham and Injury H3K27ac ChIP-seq runs and those peaks with statistically significant differential binding. C, Workflow diagram and name designations for data sets. Total H3K27ac ChIP-seq peaks are called using MOSAiCs (Sham-All and Injury-All). Differentially bound peaks are determined using DB ChIP, which identifies peaks that are significantly changed in between two conditions (Sham-DB and Injury-DB). Peak sequences were analyzed using Homer to identify enriched transcription factor motifs, and H3K27ac-marked enhancers were analyzed with respect to previous ChIP-seq data sets of transcription factor binding in peripheral nerve (Egr2 and Sox10). Genes linked to differential peaks were correlated with published nerve injury expression microarray data. DAVID was used to determine gene ontology groupings for genes determined to be regulated by DB sites.

Figure 3. Analysis of ShamDB peaks. A, H3K27ac profiles in Sham and Injury conditions are shown for ShamDB peaks in genes downregulated during nerve injury: Egr2 and Hmgcr. B, In vivo ChIP H3K27ac assays were performed for sites with H3K27ac loss after nerve injury. Independent pools of chromatin from Sham or Injured sciatic nerve were immunoprecipitated using an anti-H3K27ac antibody and negative control antibody (IgG). ChIP samples were analyzed using quantitative PCR with the indicated primer sets and percent recovery is calculated relative to input. Primer locations for the Drp2 and Mag genes are the single ShamDB peaks, shown in Figures 2A and 5A. C, Intersection of annotated Sham or Injury differentially bound peaks with genes induced or repressed 2 fold or greater after nerve injury (23). D, Transcription factor binding motifs generated from Sham-DB peaks. The percentage indicates the proportion of Sham-DB peaks with indicated motif, and the p-value shows the significance of motif association relative to a randomized set of peaks. Figure 4. Gene ontology for Sham peaks colocalizing with Egr2/Sox10 binding. A, The gene ontology cluster and Kegg pathway enrichment calculated for genes annotated to shared Egr2 and ShamDB or Sox10 and Sham-DB peak regions. B, The gene ontology cluster and Kegg pathway enrichment calculated for gene annotated to shared Egr2 and Sham-All or Sox10 and Sham-All peak regions. C, Tag densities for H3K27ac, Egr2, and Sox10 across Lamb1, Itga6, and Dag1 – genes important for extracellular matrix interactions and bound by Egr2, Sox10 transcription factors. Figure 5. Egr2 and Sox10 bind a small subset of developmentally important enhancers for Schwann cell myelination. A, Egr2 and Sox10 binding is shown relative to Sham-DB peaks for Sema5a, Mag, and Fa2h genes, which decrease after nerve injury. B, The heatmap plot shows enrichment of H3K27ac sequencing tags aligned to a 5kb window centered on Egr2 or Sox10 binding sites contained

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Figure 2. Dynamic regulation of H3K27ac-marked enhancer regions following nerve injury. A, H3K27 acetylation and DB tracks are shown for sham and injury conditions at Drp2 and Ngfr genes. The position of Egr2 binding peak track and a called peak within Drp2 is also shown. B, The heatmap plot shows enrichment of H3K27ac sequencing tags aligned to a 5kb window around DB peaks in sham and injury conditions. The height of the two heatmaps were adjusted to be equal, even though they represent different numbers of genes. While the heat map is centered on the DB peaks, there are sometimes adjoining regions within the 5 kb window that were also differentially regulated, and these were clustered together during heat map generation. C, The bar graph shows genomic distribution of differentially bound (DB) and Shared peak sets for intronic, intergenic, and promoter (-1kb to +0.88kb around TSS) classifications.

ChIP-seq Analysis of Peripheral Nerve Injury within DB enhancer sites. C,D Transcription factor binding motifs generated from C, Egr2 and Sham-All or D, Sox10 and Sham-All peaks. The percentage indicates proportion of Sham-All peaks with the indicated motif, and the p-value shows the significance of motif association relative to a randomized set of peaks. Figure 6. Analysis of InjuryDB peaks. A, Transcription factor binding motifs generated from Injury-DB peaks. The p-value shows the significance of motif association relative to a randomized set of peaks. B, The histogram indicates motif frequency for c-Jun and Runx2 relative to the center of Injury DB peaks. C, Immunohistochemistry was performed for Sox10 and Runx2 on sections from peripheral nerve in both sham and 3 d post-injury conditions. D, Expression of c-Jun target genes was determined by quantitative RT-PCR after siRNA-mediated reduction of c-Jun, or E) Runx 2 in primary Schwann cells relative to a negative control siRNA. Error bars represent standard deviation and asterisks denote p-value (* ≤ 0.05, ** ≤ 0.005). The statistics were generated from independently generated samples in triplicate (n = 3).

Figure 8. cJun binds a small subset of developmentally important enhancers for Schwann cell repair. A, ChIP-seq profiles for sham and injury conditions and InjuryDB peaks are shown at Shh, Hmga1, and Gdnf genes. B, In vivo ChIP-qPCR analysis of selected Injury-DB peaks was performed as described for Figure 7 using an anti-H3K27ac antibody and negative control antibody (IgG). ChIP samples were analyzed using quantitative PCR with the indicated primer sets and percent recovery is calculated relative to input. C, ChIP assays for c-Jun binding was performed at the indicated primer locations in Sham and Injury chromatin. Error bars represent standard deviation and asterisks denote significant p-value (* ≤ 0.05, ** ≤ 0.005).

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Figure 7. Injury-induced regulatory elements in the Runx2 gene. A, ChIP-seq profiles for H3K27ac with Sham-DB and Injury-DB peak designations are shown for the Runx2 gene. Primers for ChIP-qPCR validation are marked at InjuryDB peaks. B, c-Jun or Runx2 binding sites found at each Injury-DB peak are listed. C, Chromatin from Sham or Injured sciatic nerve were immunoprecipitated using an antiH3K27ac antibody and negative control antibody (IgG). ChIP samples were analyzed using quantitative PCR with the indicated primer sets and percent recovery is calculated relative to input. D, Similar ChIP assays were performed for c-Jun binding to the indicated sites within Runx2. Error bars represent standard deviation and asterisks denote significant p-value (* ≤ 0.05, ** ≤ 0.005).

ChIP-seq Analysis of Peripheral Nerve Injury

Table 1. ChIP Primers Gene Primer

Forward

Reverse

rHMGA1-DB Ch1

1

GTGGAGGGTGGGTTAAGGAT

TTCTGAGAGCCCAGTGAGGT

rmHMGA1-TFs Ch1

2

CTGCTGAGTCACCCACACAC

GAGATGCCCTCCTCTTCCTC

rShh-Jun Ch4

1

GCCACTTACTGGCCTACCAT

TCAGACCGACCAAGCTCTTT

rShh-DB Ch1

2

ATGTTAATGCAGGTGCCACA

GGGTTCCAGTGCTTTGTCAT

rRunx2-Tf Ch1

1

TGCAGAGTTCTGCTCTCCAA

ACAAAAGGCTTGTGGTGAGG

rRunx2-Tf Ch2 mrRunx2-RUNX2 Ch4 mrRunx2-RUNX2 Ch5

2

GTGAGGATGGCAGATGTCCT

ACCTCATCAAAACCCCACAA

3

CCACCCAGCTGCTTGTACTT

TTCACATTCACTGCCCTCAG

4

AAGCCACAGTGGTAGGCAGT

TTGTTTGTGAGGCGAATGAA

Gdnf

rGDNF-Jun Ch1

1

CCATGAATCGGGAGTAGGAA

CCGGTCAAAGAGCACAAACT

Egr2

rEGR2 pro Ch1

1

GAAAGTCTCGGAGAACCGGAA

GCAGCGATGGTAGCTCTGTCT

Hmga1 Shh Runx2

mrEGR2 MSE

2

TCCTGACCAGAAAGATTGTTA

TGCAGGATTTCAGCTTTGTGA

Hmgcr

rmHMG pro CRE Ch1

1

TCGTGACGTAGGCCGTCAG

CCAATAAGGAAGGATCGTCCG

Mag

rMag Int Ch2

1

ACAGGGATCTTTTGTATTGGCTACA

CGCAGCAGGCTTAGGGTG

Pmp22

rPmp22 Ch24

1

CCTTCCTCCTGTGCAGCATAAG

GGGCCTGGCGAAGCA

mrPmp22 -90kb Ch2

2

TCACTCCCACTGGCTGGAA

AAATGCCCTTTGTCCGAACTC

mrTEKT3 Ch1 (Neg.)

Neg

AATACCAGCAGATCCGGAAGAC

TTGACTCGTTCTCCCAGGTTTT

AGGCCCGCCCTCCTGCACACG

TTTGCGTCGCTGCAGAAG

rPmp22 -2 Ch1

3

Table 2: qRT-PCR Primers Gene

Primer

Forward

Reverse

18S

18S rRNA

CGCCGCTAGAGGTGAAATTCT

CGAACCTCCGACTTTCGTTCT

cJun

rJun TM2

GAGAGGAAGCGCATGAGGAAC

CCTTTTCCGGCACTTGGAG

Runx2

rRunx2 TM

GCACCCAGCCCATAATAGAA

TGGAGATGTTGCTCTGTTCG

Shh

rShh TM

GCGGGCATCCACTGGTACT

TCGGACTTCAGCTGGACTTGA

Olig1

rOlig1 TM

ACATCAAGGGTGTTGCCGAT

GACACCGGACTCTGGGCT T

GDNF

rGDNF TM

ACTGACTTGGGTTTGGGCTA

CCTGGCCTACCTTGTCACTT

HMGA1

rHMGA1 TM2

AGTGCAGGGGAAGCTTAGGT

GCTTGATGGGGGACATACAT

DRP2

rDrp2 TM

GAGAAGATCCTGGCCCATTT

CCTCAGCTCTCCCTGAAGAA

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#

ChIP-seq Analysis of Peripheral Nerve Injury

Egr2, Sox10, and H3K27ac overlapping regions Egr2-Sox10 composite sites Egr2 and ShamAll Sox10 and ShamAll Egr2-Sox10 and ShamAll Egr2 and ShamDB Sox10 and ShamDB Egr2-Sox10 and ShamDB Egr2 and InjuryAll Sox10 and InjuryAll Egr2-Sox10 and InjuryAll Egr2 and InjuryDB Sox10 and InjuryDB Egr2-Sox10 and InjuryDB

Peaks 618 2679 2047 454 312 312 94 1891 1281 304 44 51 3

Relative to:

%TF

37.32% Egr2 peaks 34.75% Sox10 peaks 73.46% Egr2/Sox10 peaks 11.65% Egr2/ Sham-All 15.24% Sox10/Sham-All 20.70% Egr2/Sox10/Sham-All 26.34% Egr2 peaks 21.75% Sox10 peaks 49.19% Egr2/Sox10 peaks 2.33% Egr2/Injury-All 2.49% Sox10/Injury-All 0.49% Egr2/Sox10/Injury-All

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Table 3: Peak overlap frequency between Egr2, Sox10, and H3K27ac overlapping regions. Values indicated number and overlap percentage between Egr2, Sox10, and differentially bound H3K27ac peaks (ShamDB or InjuryDB) or total H3K27ac peaks at each condition (ShamAll or InjuryAll). Percentages are calculated from fraction of all sites shared for each condition.

A

B Sha m/Sciatic nerve tra ns ection s urgery

Injury

MOSAiCS 28,114 pea ks 31,894 pea ks Sha red DB ChIP 13,298

72hrs

Sham-DB

3,290 peaks

Injury-DB

4,312 peaks

MOSAiCs

Ini tial peak calling for ea ch ChIP-seq set Sha m-All Injury-All Multi-Dimensional Analysis ChIP-seq DAVID Microarray HOMER

Figure 1

DB ChIP

Di fferential peak determination between conditions Sha m-DB Injury-DB

Egr2 and Sox10 ChIP-seq Gene Ontology Nerve injury Gene Expression Motif Analysis

Active enhancer and tra ns cription factor i dentification

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H3K27a c Sham ChIP H3K27a c Injury ChIP

C

Sham

A ShamDB

Drp2

5kb

Ngfr

10kb

20

Sham K27ac InjuryDB

1 20

Injury K27ac

1 25

Egr2

1

35

Sham K27ac

1

InjuryDB 35 Injury K27ac

B

1

Sha m DB Sham

Injury

Injury DB Sham

Injury

C 100% 80%

H3K27ac

60% 40% 20% 0% Shared Peak center -2.5kb

Figure 2

+2.5kb

ShamDB

Intergenic Promoter

InjuryDB Intron Exon

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ShamDB

B

10kb

Drp2

C

D

Nerve Injury Expression

Mag

HMGCR

Egr2

Figure 3

Injury-DB

No Overlap

Injury

Control

Sham-DB Motif % Peaks

Sham-DB

Sham

Injury

* *

Sham

Injury

* *

Sham

1.0 0.0

*

* *

Injury

Egr2-MSE

2.0

p-value

Tead (TEA)

35.6%

1e-18

Sox (HMG)

45.3%

1e-13

Egr (Zf)

25.4%

1e-8

NF1 (CTF)

59.6%

1e-8

Nur77(NR)

11.1%

1e-4

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Injury K27ac

3.0

Sham

InjuryDB

4.0

Injury

Sham K27ac

HMGCR

K27Ac_H3

5.0

ShamDB H3K27a c

IgG

6.0

Sham

5kb

% Recovery

A

A

B

Egr2 + Sham-All Ontology Count

Egr2 + Sham-DB Ontology

p-value

Axon ensheathment

19

5

Positive reg of gene expression

80

Reg of locomotion

Count

p-value

Reg of transmission of nerve impulse

10

6 E-04

1 E-08

Axon ensheathment

5

4 E-03

33

1 E-05

Protein tyrosine phosphatase activity

6

5 E-03

Vasculature development

39

8 E-06

Response to carbohydrate stimulus

6

7 E-03

Kegg pathway: Focal Adhesion

34

3 E-05

Kegg: Neurotrophin signaling pathway

7

7 E-03

E-09

Sox10 + Sham-DB Ontology Count

p-value

Count

p-value

Positive reg of transcription

67

4 E-10

Reg of cell morphogenesis

8

3 E-03

Reg of action potential in neuron

19

1 E-07

Positive reg of synaptic transmission

5

2 E-03

Neuron projection development

41

9 E-07

Reg of neuron differentiation

9

3 E-03

Reg of nervous system development

35

4 E-06

Protein oligomerization

10

3 E-03

Kegg pathway: Adherens junction

23

9 E-06

Kegg pathway: Axon guidance

7

1 E-02

C

Itga6

Lamb1

H3K27ac Egr2 Sox10

35

30

1

1

30

55

1 30

1 55

1

1

Dag1 H3K27ac Egr2 Sox10

30 1 25 1 25 1

Figure 4

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Sox10 + Sham-All Ontology

A

Sema5a ShamDB

25

InjuryDB

25

100kb

B H3K27Ac

Injury K27ac 1

Sox10

Sox10-DB

Sham Injury Sham Injury

Sham K27ac 1

Egr2

Egr2-DB

25 1 25

Peak center

1

Mag ShamDB

20

InjuryDB

20

5kb

C Egr2 + H3K27ac Motif

Sham K27ac 1 Injury K27ac 1 Egr2 Sox10

22 1 22 1

Fa2h ShamDB

Sham K27ac InjuryDB

Injury K27ac Egr2 Sox10

30 1 30

10kb

+2.5kb

%Peaks

p-value

Egr2(Zf)

19.0%

1 E-26

TEAD(TEA)

41.1%

1 E-12

Sox(HMG)

41.2%

1 E-09

Maz(Zf)

59.7%

1 E-06

CRE(bZIP)

12.8%

1 E-05

D Sox10 + H3K27ac Motif

%Peaks

p-value

Sox(HMG)

61.3%

1 E-53

1

TEAD(TEA)

43.0%

1 E-10

40

NF1-halfsite(CTF)

64.5%

1 E-06

1 40

GATA-IR4(Zf)

6.0%

1 E-05

Foxo1(Forkhead)

71.6%

1 E-04

1

Figure 5

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-2.5kb

A

B %Peaks

p-value

Jun-AP1 (bZIP)

29.3%

1e-278

ETS1(ETS)

51.8%

1e-36

RUNX(Runt)

41.1%

1e-28

ELF5(ETS)

39.7%

1e-26

Injury

0.0005 0.0000

0

Jun

2000

Runx2

siNeg 1.0 0.5

25μm

* *

* *

siJun

*

* *

* *

0.0

E

Merge

RUNX

1.5 1.0 0.5 0.0

siNeg * *

siRunx2 * *

*

* *

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D

Sox10

Figure 6

0.0010

Injury DB Peak Apex

Normalized Expression

Sham

0.0015

-2000

Normalized Expression

C

Motif per bp per peak

Injury-DB Motif

A

Runx2 ShamDB Sham K27ac InjuryDB Injury K27ac

20 1 20 1

1

Pri mer:

B

% Recovery

4

2

3

4

Primer:

1

2

3

4

cJun

GTGACTCA

ATGAGTCAC

N/A

N/A

Runx2

GTGTGGTCT

ATTGTGGTC

TTGTGGTGA

TTGTGGGTGA

Rabbit IgG

3

K27Ac_H3

* *

* *

*

* *

2 1 0 Sham Injury Sham Injury Sham Injury Sham Injury Sham Injury 1

2

3

4

Negative

Runx2

Control

D % Recovery

0.8

IgG

cJun

*

0.6 *

0.4 0.2 0

Sham Injury Sham Injury Sham Injury Sham Injury Sham Injury 1

2

3 Runx2

Figure 7

4

Neg

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C

20kb

A

B

Shh

% Recovery

10kb

ShamDB 25 Sham K27ac 1 InjuryDB 25 Injury K27ac 1

6

IgG

*

K27Ac_H3

*

4

*

2

Hmga1

1kb

1

2

1

Shh

ShamDB 25 Sham K27ac 1 InjuryDB 25 Injury K27ac 1

2 HMGA1

Injury

Sham

Injury

Sham

Injury

Sham

Injury

Sham

Injury

2

Sham

1

Sham

Pri mer:

Injury

0

1

Neg

GDNF

Control

C

Pri mer:

Figure 8

2 Shh

1

2 Hmga1

Injury

Sham

Injury

Sham

Injury

Sham

Injury

Sham

Injury

0

1

1

*

* Sham

ShamDB 30 Sham K27ac 1 InjuryDB 30 Injury K27ac 1

0.5 Injury

5kb

cJun

1

Sham

GDNF

2

% Recovery

1

Pri mer:

IgG

*

1.5

1 GDNF

Neg Control

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2

Gene Regulation: Dynamic Regulation of Schwann Cell Enhancers after Peripheral Nerve Injury Holly A. Hung, Guannan Sun, Sunduz Keles and John Svaren J. Biol. Chem. published online January 22, 2015

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Access the most updated version of this article at doi: 10.1074/jbc.M114.622878

Dynamic regulation of Schwann cell enhancers after peripheral nerve injury.

Myelination of the peripheral nervous system is required for axonal function and long term stability. After peripheral nerve injury, Schwann cells tra...
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