(a) Cultures of postnatal DRG neurons and whole cerebellum were used to generate cDNA libraries representing the respective mRNA populations. The cerebellum library driver was then hybridized to the DRG library tester in great excess, followed by recovery of non-bound DRG cDNAs by HAP chromatography. The resulting “subtracted” library was sequenced and BLASTed against the UniGene, whereupon 1,068 unique genes were identified. For the microarray analysis, a data set consisting of 5 replicates of LCM DRG neurons from adult mice was obtained from GEO and compared to our own set of 3 replicates of P11 cerebellum that was collected on the same microarray platform (MG-U74Av2). (b) DAVID was used to identify gene ontology terms describing subtraction genes. Grey bars represent the number of subtraction genes with the labeled annotation, and the solid line denotes the Benjamini-Hochberg corrected EASE p-value. Only terms with a corrected p-value less than 0.05 were considered.

(a) Model demonstrating how differentially active TFs may be detected by the analysis of DRG enriched gene promoters. In PNS neurons, an active TF (marked by *) is able to bind target sequences, inducing the transcription of mRNAs that can be detected by the Subtraction and Microarray. In CNS neurons, a TF may be unable to drive expression of target sequences due to a lack of upstream signals or co-activators, the presence of binding proteins dampening activity (BP), or an altered genomic landscape. (b) Venn Diagram of 132 transcription factor binding consensuses found to be significantly overrepresented in the promoter regions of DRG enriched genes. 1200 bp promoter regions (1000bp upstream and 200 bp downstream of TSS) were scanned for consensus binding sites from TRANSFAC 10.2 and from a paper by Xie et al. (2005). (c) 28 TF binding consensuses from TRANSFAC and 15 from Xie et al. (2005) were found to be significantly overrepresented in both the Subtraction and the Microarray data sets. Significance was determined by a two-tailed Fisher’s Exact Test, followed by a Benjamini-Hochberg correction for multiple comparisons.

(a) Western blot of COS7 cells transfected with GFP and full length STAT3 cDNA stained for total STAT3 and βIII-tubulin. (b) Confocal micrograph showing a single cerebellar granule neuron transfected with the STAT3 construct. After transfection, cultures were probed with antibodies for βIII-tubulin (red) and total STAT3 (green). Scale bar: 50µm. (c) Mean total neurite length of CGNs transfected with GFP, wild type STAT3 and constitutively active STAT3-C, and grown on poly-D-lysine for 48 hours. Only STAT3-C significantly improved outgrowth. At least 100 STAT3 positive neurons were quantified in each experiment, n=4. Error bars = 95% confidence intervals. Significance determined by one way ANOVA followed by Dunnett’s post-hoc test, ** = p<0.01. (d) Overexpression of a constitutively active form of IRF1, IRF1-VP16, increased mean total neurite outgrowth compared to the wildtype form of the gene. IRF1 is a major downstream target of STAT3. Significance was determined by an unpaired two-tailed Student’s t-test, * = p<0.05.

(a) Metacore generated screenshot of nearest neighbors network for transcription factor BHLHE40. Each node represents a protein or gene, and connecting arrows represent a curated interaction between the two nodes. Outward arrows denote regulation by BHLHE40, whereas inward arrows denote regulation of the factor. (b) Venn Diagram of transcription factor networks found to be significantly overrepresented in DRG neurons by Metacore. This analysis considered all known TFs within Metacore and was not biased towards the expression level of the TFs. Using this approach, we expect to identify differentially active TFs in addition to factors that may have been missed by the Subtraction or Microarray. (c) 30 TF networks were found to be significantly overrepresented in both the Subtraction and the Microarray data sets. P-values were determined by a method developed by Metacore (discussed in the methods section), followed by a Benjamini-Hochberg correction for multiple comparisons.

(a–b) Representative Western blot of lysates from P7-10 mouse dorsal root ganglia, cerebellum, and cerebral cortex probed for (a) total STAT3 and (b) Y705 phosphorylated STAT3. Neuronal specific βIII-tubulin was used as a loading control. (c–e) STAT3 co-localizes with NeuN but not Hoechst positive Schwann cells in the nerve root. (c) NeuN, (d) total STAT3, (e) the nuclear dye Hoechst. (f–h) High magnification confocal micrograph of (f) NeuN, (g) total STAT3, and the (h) merged image showing co-localization. (i) DRG neurons express nuclear Y705 phosphorylated STAT3 constitutively. (j–l) High resolution confocal micrographs showing (j) Islet 1 staining neuronal nuclei, (k) Y705 STAT3 and (l) the merged image. Scale bars: (c,d,e,i), 100µm; (f,g,h,j,k,l), 10µm. (m) The relative expression of 84 JAK-STAT pathway members in P7-10 dorsal root ganglia and cerebellum was probed using real-time PCR. Multiple factors that regulate and are regulated by STAT3 activity were found to be enriched in DRGs, suggesting the presence of at least one PNS specific STAT3 signaling pathway. (n) Distribution of the STAT consensus binding motif (TCCCRGAAR) within the promoter regions of 126 DRG enriched genes. Individual STAT consensus matches for genes identified by the subtraction (green hatches) and microarray (red hatches) are shown below. Many sites cluster within 200bp upstream of the transcription start site (TSS).