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Loss-of-function mutations in the Src homology 3 (SH3) domain and tetratricopeptide repeats 2 (SH3TC2) gene cause autosomal recessive demyelinating Charcot–Marie–Tooth neuropathy. The SH3TC2 protein has been implicated in promyelination signaling through axonal neuregulin-1 and the ERBB2 Schwann cell receptor. However, little is known about the transcriptional regulation of the SH3TC2 gene. We performed computational and functional analyses that revealed two cis-acting regulatory elements at SH3TC2—one at the promoter and one ∼150 kb downstream of the transcription start site. Both elements direct reporter gene expression in Schwann cells and are responsive to the transcription factor SOX10, which is essential for peripheral nervous system myelination. The downstream enhancer harbors a single-nucleotide polymorphism (SNP) that causes an ∼80% reduction in enhancer activity. The SNP resides directly within a predicted binding site for the transcription factor cAMP response element binding protein (CREB), and we demonstrate that this regulatory element binds to CREB and is activated by CREB expression. Finally, forskolin induces Sh3tc2 expression in rat primary Schwann cells, indicating that SH3TC2 is a CREB target gene. These findings prompted us to determine if SNP genotypes at SH3TC2 are associated with differential phenotypes in the most common demyelinating peripheral neuropathy, CMT1A. Interestingly, this revealed several associations between SNP alleles and disease severity. In summary, our data indicate that SH3TC2 is regulated by the transcription factors CREB and SOX10, define a regulatory SNP at this disease-associated locus and reveal SH3TC2 as a candidate modifier locus of CMT disease phenotypes.
Charcot–Marie–Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited peripheral neuropathies that affect motor and sensory neurons. Clinical presentations include muscle wasting, foot deformities and impaired sensation (1). Mutations in the Src homology 3 (SH3) domain and tetratricopeptide repeats 2 (SH3TC2) gene cause autosomal recessive CMT type 4C (CMT4C) (2). CMT4C is the most common form of autosomal recessive demyelinating CMT disease, accounting for up to 26% of all cases (2–5). As with other subtypes of CMT disease, patients with CMT4C show variability of disease onset and severity (2,4). For example, onset of disease can vary from 2 to 50 years of age and the motor phenotype varies from mild gait disabilities to impairment requiring canes or wheelchairs. Furthermore, ∼60% of patients with CMT4C have scoliosis (2–4). Thus, there are likely genetic and environmental factors that modify the CMT4C phenotype.
SH3TC2 is expressed specifically in Schwann cells (6) and encodes a protein with SH3 and tetratricopeptide domains important for protein–protein interactions. SH3TC2 localizes to the endocytic recycling pathway (6–9) and interacts with the GTPase Rab11, a known regulator of recycling endosomes. Interestingly, mutant forms of SH3TC2 are unable to associate with Rab11 in vitro, suggesting that disease-associated SH3TC2 mutations affect the rate of endosome recycling (8,9). It was recently shown that SH3TC2 plays a role in neuregulin-1 (Nrg1)/ERBB signaling, which is critical for the proliferation and migration of Schwann cells and the subsequent myelination of peripheral nerve axons (10). Specifically, SH3TC2 interacts with and internalizes ERBB2 and depletion of SH3TC2 in vivo results in downregulation of key ERBB targets (11). Indeed, two CMT4C-associated missense mutations that map to the interaction domain prevent internalization of ERBB2.
Over 30 SH3TC2 mutations have been identified in patients with CMT4C in either a homozygous or compound heterozygous state. The majority of the mutations act via a loss-of-function mechanism and disease-associated alleles include nonsense, missense and splice-site mutations (2,3,12–16). Despite the loss-of-function nature of known pathogenic variants, regulatory mutations (e.g. those in promoters or enhancers) have not been identified at SH3TC2. Interestingly, the identification of patients with a CMT4C phenotype that are heterozygous for a single SH3TC2 coding mutation (15,16) suggests that mutations at a second locus or mutations in non-coding, transcriptional regulatory elements at SH3TC2 account for a certain portion of CMT4C disease.
Currently, little is known about the transcriptional regulation of SH3TC2. Addressing this issue will be important for fully understanding the biology of the SH3TC2 locus and for identifying the full spectrum of disease-associated SH3TC2 mutations. This information will also assist the identification of functional polymorphisms or modifiers that, by altering SH3TC2 gene expression, may contribute to the variable clinical phenotype observed in patients with CMT4C or other CMT subtypes with a myelin-based pathology, such as the most common form of CMT disease: CMT1A caused by duplication of the peripheral myelin protein 22 (PMP22) (17–19). Here, we employ computational and functional analyses to identify transcriptional regulatory elements at SH3TC2 and report the characterization of the SH3TC2 promoter and a downstream enhancer. Interestingly, the latter element harbors a common single-nucleotide polymorphism (SNP) that dramatically decreases regulatory function. These findings provide key information regarding the biology of the SH3TC2 locus and reveal candidate sequences for mutations and modifiers of CMT disease.
SH3TC2 harbors seven putative transcriptional regulatory elements
Multiple-species comparative sequence analysis is a powerful tool for predicting cis-acting transcriptional regulatory elements (20). To identify evolutionarily conserved sequences at SH3TC2, we aligned genomic sequences spanning SH3TC2 and extending to the flanking loci (ADRB2 and ABLIM3) from human, mouse, rat, dog, cow and opossum using MultiPipMaker software (21). Next, we identified non-coding, non-repetitive genomic sequences within the alignment that are at least 5bp long and identical among all six species using ExactPlus (EP) (22). This analysis revealed 49 genomic segments ranging from 5 to 19 bp in length. Importantly, these smaller fragments fell into seven clusters of multiple-species conserved sequences (MCSs)—one directly upstream of the predicted transcription start site (TSS) of SH3TC2, one within the 3′ untranslated region and five downstream of SH3TC2 (Table 1 and Fig. 1A). We considered these seven genomic segments to be candidate transcriptional regulatory elements for SH3TC2.
|Element ID
|Genomic locationa
|Size (bp)
|chr5:148 422 779–148 423 445
|chr5:148 188 297–148 188 683
|chr5:148 189 329–148 189 715
|chr5:148 192 623–148 193 023
|chr5:148 203 383–148 203 846
|chr5:148 272 774–148 273 158
|chr5:148 346 393–148 346 955
|Element ID
|Genomic locationa
|Size (bp)
|chr5:148 422 779–148 423 445
|chr5:148 188 297–148 188 683
|chr5:148 189 329–148 189 715
|chr5:148 192 623–148 193 023
|chr5:148 203 383–148 203 846
|chr5:148 272 774–148 273 158
|chr5:148 346 393–148 346 955
aCoordinates are from the March 2006 UCSC Human Genome Browser (hg18).
The SH3TC2 promoter and SH3TC2-MCS5 direct luciferase expression in cultured Schwann cells
Our computational analyses revealed seven conserved genomic regions at the SH3TC2 locus that potentially harbor transcriptional regulatory elements. To determine if these genomic segments have regulatory activity in relevant cells in vitro, we tested the ability of each to direct luciferase reporter gene expression in cultured Schwann (S16) cells. S16 cells are an immortalized rat Schwann cell line that exhibit a high transfection efficiency and express many myelin-related genes (e.g. PMP22, MPZ, MBP and MAG) and Schwann cell transcription factors (e.g. SOX10 and EGR2). Indeed, in a comparative analysis of gene expression in multiple Schwann cell lines, S16 cells were the most similar to myelinating Schwann cells (23). Briefly, each genomic segment (Table 1) was cloned upstream of a minimal promoter directing the expression of a luciferase reporter gene and each resulting construct was transfected into S16 cells. Luciferase activity was then measured compared with a control vector with no genomic insert ('Empty' in figures). To identify the most promising regulatory elements at SH3TC2, a 10-fold or higher increase in luciferase activity was considered indicative of 'strong' enhancer activity. SH3TC2-MCS1, MCS2, MCS3, MCS4 and MCS6 did not display 'strong' enhancer activity in Schwann cells (<5-fold each; Fig. 1B); however, SH3TC2-MCS1 (P-value 0.003) and SH3TC2-MCS4 (P-value 2.2 × 10−8) did significantly increase reporter gene expression to a lesser degree (2.1- and 3.9-fold, respectively). In contrast, the SH3TC2 promoter and SH3TC2-MCS5 demonstrated a 30-fold (P-value 8.74 × 10−6) and 13-fold (P-value 2.34 × 10−7) increase in activity, respectively, in these cells (Fig. 1B). To determine if the enhancer activities of the SH3TC2 promoter and SH3TC2-MCS5 are specific to Schwann cells, each genomic segment at SH3TC2 was reevaluated in a non-glial cell line—immortalized mouse motor (MN-1) neurons. Importantly, transcription factors important for Schwann cell function (e.g. SOX10) are not expressed in MN-1 cells (24). None of the regions tested displayed 'strong' enhancer activity in MN-1 cells. Indeed, the mean fold-induction of each of the seven tested genomic segments was <5 (Fig. 1C). Combined, these data suggest that the SH3TC2 promoter and SH3TC2-MCS5 are important for transcriptional regulation of SH3TC2 in Schwann cells.
The SH3TC2 promoter harbors consensus sequences for Schwann cell transcription factors
The SH3TC2 promoter studied here encompasses 667 bp upstream of the initiation codon (Fig. 2A). To characterize the position of the SH3TC2 TSS in Schwann cells, we performed 5′ rapid amplification of cDNA ends (5′RACE). Briefly, Sh3tc2 cDNA was generated from RNA isolated from cultured rat Schwann (S16) cells and 5′RACE was performed using a reverse primer in exon 4 of the rat Sh3tc2 locus. The resulting PCR product was sequenced and aligned to rat genomic DNA sequences. These studies revealed a single Sh3tc2 TSS located 37 bp upstream of the start codon in rat Schwann cells (Fig. 2A–C). This position also corresponds to 37 bp upstream of the initiating methionine codon at the human SH3TC2 locus and differs from the current annotation of the SH3TC2 RefSeq mRNA on the University of California at Santa Cruz (UCSC) Human Genome Browser (Fig. 2A).
SH3TC2 is specifically expressed in Schwann cells (6), suggesting that the locus is regulated by transcription factors important for these cells. To address this, we employed the TRANSFAC prediction algorithm (25) to search the SH3TC2 promoter for the consensus sequences of three transcription factors critical for Schwann cells: SOX10, EGR2 and POU3F2 (26). These analyses revealed six SOX10 consensus sequences (bold text in Fig. 2C), one POU3F2 consensus sequence (red text in Fig. 2C) and one EGR2 consensus sequence (blue text in Fig. 2C). Note that the POU3F2 consensus sequence overlaps with one of the SOX10 consensus sequences. SOX10 is known to function as either a monomer or a dimer (22,27). As a monomer, SOX10 binds to a single SOX10 consensus sequence and as a dimer it binds to two consensus sequences oriented in a head-to-head fashion. Two of the SOX10 consensus sequences are consistent with binding to monomeric SOX10 (SOX10 A and SOX10 B in Fig. 2C) and four of the SOX10 consensus sequences cluster into two head-to-head sequences consistent with binding to dimeric SOX10 (SOX10 C and SOX10 D in Fig. 2C). Note that the 'TTTGA' monomer (bold, red text in Fig. 2C) in the SOX10 C dimeric consensus sequence was not initially detected by our computational analyses (see Materials and Methods for details). However, we considered SOX10 C a potential head-to-headbinding site due to the preferential 6 bp spacing (27) of the two monomeric predictions (Fig. 2C and D) and the conservation of the entire prediction among diverse mammalian species (Fig. 2D). Interestingly, reducing SOX10 function decreases the expression of Sh3tc2 in Schwann cells (28,29) and SOX10 binding was previously detected at the SH3TC2 promoter region by ChIP-Seq analysis of nuclei isolated from rat sciatic nerves (28) (also shown in Supplementary Material, Fig. S1A). The ChIP-Seq data directly overlap the SOX10 consensus sequences described above; however, the precise sequences responsible for SOX10 binding have not been identified.
SOX10 consensus sequences are necessary for the activity of the SH3TC2 promoter
The presence of conserved SOX10 consensus sequences within the SH3TC2 promoter suggests that these nucleotides are necessary for the observed regulatory activity in Schwann cells (Fig. 1B). To test this, we deleted or mutated each consensus sequence and tested the ability of the resulting genomic segment to direct luciferase reporter gene expression in cultured S16 cells compared with the wild-type SH3TC2 promoter. For deletion analysis, the entire consensus sequence was removed from the construct ('Δ' in Fig. 3). For mutation analysis, the consensus sequence was mutagenized to complement but not reverse the sequence ('Mut' in Fig. 3). This should ablate the binding capabilities of the transcription factor while retaining the overall GC content of the genomic segment. Because SOX10 A and SOX10 B are palindromic (Fig. 2C) binding to SOX10 may not be disrupted by mutagenesis—we therefore only deleted these consensus sequences. Finally, deletion of the POU3F2 consensus sequence would also delete SOX10 C—we therefore mutated the 'GCAGCC' nucleotides between the two SOX10 consensus sequences in SOX10 C to only disrupt POU3F2 binding (Fig. 2C). Deletion of the SOX10 A and SOX10 B consensus sequences and mutagenesis of the POU3F2, EGR2 and SOX10 D consensus sequences did not significantly reduce the activity of the SH3TC2 promoter (Fig. 3A). In contrast, mutagenesis and deletion of the SOX10 C consensus sequence resulted in an ∼60% (P-value 1.67 × 10−7) and ∼50% (P-value 8.11 × 10−7) reduction in enhancer activity, respectively, compared with the wild-type SH3TC2 promoter (Fig. 3A). These data indicate that this SOX10 consensus sequence is required for the observed activity of the SH3TC2 promoter.
Visual examination of the SH3TC2 promoter at the UCSC Human Genome Browser revealed the presence of an SNP (rs7705960) with a minorallele frequency of 0.442 in individuals of European descent from the HapMap dataset. Since SNPs within transcriptional regulatory elements can affect gene transcription rates, we compared the enhancer activities of the major and minor alleles of rs7705960 in cultured Schwann cells in the context of the SH3TC2 promoter (Table 1). These efforts revealed that the enhancer activity of the minor-allele harboring genomic segment (Fig. 3A) was similar to the segment carrying the major allele ('SH3TC2-Promoter' in Fig. 3A), indicating that this SNP does not affect the activity of the SH3TC2 promoter.
SOX10 induces the activity of the SH3TC2 promoter
Disrupting the head-to-head SOX10 Cbinding site causes a reduction in SH3TC2 promoter activity, suggesting that SOX10 acts upon this element in Schwann cells. To determine if SOX10 induces the activity of the SH3TC2 promoter we employed a SOX10 overexpression assay in SOX10-negative MN-1 cells. Briefly, a construct harboring the SH3TC2 promoter upstream of a luciferase reporter gene was transfected into MN-1 cells with or without a construct harboring a CMV promoter directing wild-type SOX10 expression. In the presence of wild-type SOX10, the activity of the SH3TC2 promoter dramatically increased (∼28-fold, P-value 7.36 × 10−10; Fig. 3B).
If SOX10 is required for inducing SH3TC2 promoter activity, then suppressing endogenous SOX10 activity in Schwann cells should cause a decrease in SH3TC2 promoter activity. Certain mutations in the human SOX10 gene (in the heterozygous state) result in a dominant-negative effect by disrupting the function of the wild-type SOX10 protein (30). We analyzed the activity of the SH3TC2 promoter in the context of overexpressed, dominant-negative (E189X) SOX10 in S16 Schwann cells. Expression of dominant-negative SOX10 reduced the activity of the SH3TC2 promoter by ∼73% (P-value 1.06 × 10−5; Fig. 3C). Importantly, these data are consistent with studies showing that impairing SOX10 function reduces the expression of Sh3tc2 in rat Schwann cell models (28,29). Combined, our studies indicate that SOX10 regulates the SH3TC2 promoter via the SOX10 C binding site.
The minor allele of rs17539452 reduces SH3TC2-MCS5 activity in Schwann cells
Polymorphisms in cis-acting transcriptional regulatory elements can impact gene transcription and may be relevant for clinical phenotype variability. Examination of SH3TC2-MCS5 on the UCSC Human Genome Browser revealed that this genomic segment harbors six SNPs with a minorallele frequency of ≥0.01 (Fig. 4A). To determine if these SNPs alter the activity of SH3TC2-MCS5 in Schwann cells, we obtained four SH3TC2-MCS5 haplotypes via PCR amplification of pooled human DNA samples (Hap 1–4 in Fig. 4B). Hap 1 contains the major alleles of all six SNPs and represents the genomic segment tested in the initial luciferase assays (Fig. 1B). Hap 2 carries the minor alleles of two SNPs (rs12519780 and rs10875641) and corresponds to the reference sequence in the UCSC Human Genome Browser (hg19). Hap 3 carries the minor allele of rs12519780, and Hap 4 carriers the minor alleles of three SNPs: rs12519780, rs114059937 and rs17539452. Each haplotype was tested for the ability to direct luciferase reporter gene expression in S16 cells relative to Hap 1. The activity of SH3TC2-MCS5 Hap 2 and Hap 3 was not significantly decreased compared with Hap 1 (Fig. 4C). In contrast, the activities of Hap 4 was decreased by ∼80% (P-value 1.13 × 10−8; Fig. 4C), and a decrease in activity were observed in both the forward and reverse orientation of the enhancer (Supplementary Material, Fig. S2). These data suggest that the alleles of SNPs rs12519780, rs114059937 and rs17539452 modify the enhancer activity of SH3TC2-MCS5.
To identify the SNP(s) responsible for the decrease in SH3TC2-MCS5 Hap 4 activity, we mutagenized SH3TC2-MCS5 to harbor the minor allele of rs12519780, rs114059937 or rs17539452 alone or in various combinations (Mut A–E, Fig. 4B). Subsequently, each construct was tested for the ability to direct luciferase reporter gene expression in S16 cells relative to Hap 1. Expression constructs containing the minor alleles of rs12519780 or rs114059937 alone (Hap 3 and Mut A, respectively) or combined together (Mut C) did not exhibit significantly reduced activities compared with Hap 1. In contrast, constructs harboring the minor allele of rs17539452 alone (Mut B, P-value 1.77 × 10−6) or combined with the minor allele of rs12519780 (Mut D, P-value 1.87 × 10−9) or rs114059937 (Mut E, P-value 1.46 × 10−9) all showed at least a 70% reduction in enhancer activity in cultured Schwann cells compared with Hap 1 (Fig. 4C). These data indicate that the minor allele ('t' nucleotide) of rs17539452 is responsible for the majority of the ∼80% decrease in enhancer activity of SH3TC2-MCS5 Hap 4.
The minor allele of rs17539452 disrupts a cAMP response element binding protein (CREB) in SH3TC2-MCS5
One possible explanation for the reduced activity of SH3TC2-MCS5 Hap 4 (Fig. 4C) is that the minor allele of rs17539452 interferes with transcription factor binding. To test this, we analyzed 24 bp surrounding each allele of rs17539452 using the TRANSFAC transcription factor prediction algorithm (25). This revealed a cAMP response element (CRE) prediction for the major allele of rs17539452 that is not predicted for the minor allele (Fig. 5A and B; Supplementary Material, Fig. S3A and B). CREs are genomic elements that regulate gene transcription in response to elevated cAMP. Intracellular cAMP induces phosphorylation of the CRE-binding (CREB) protein, predominantly via the PKA pathway, which can then bind to the CRE sequence and mediate transcriptional regulation (31). CREB is a ubiquitous transcription factor expressed in S16 cells (Supplementary Material, Fig. S3C); however, tissue-restricted CREB activity has been observed in melanocytes in cooperation with SOX10 (32). We therefore computationally analyzed SH3TC2-MCS5 for SOX10 consensus sequences as described above. These efforts revealed a single SOX10 monomeric consensus sequence 7bp from the CREB consensus sequence (Fig. 5B and B). To assess the role of the CREB and SOX10 consensus sequences on the function of SH3TC2-MCS5, we generated luciferase reporter constructs harboring a deletion of the CREB consensus sequence (ΔCREB), a deletion of SOX10 consensus sequence (ΔSOX10), or a deletion of both consensus sequences (ΔCREBΔSOX10). Subsequently, each construct was transfected into S16 cells and enhancer activities were compared with SH3TC2-MCS5 Hap 1. All three deletions resulted in at least a 70% reduction in luciferase activity (P-values 2.25 × 10−8, 4.68 × 10−8 and 1.91 × 10−8, respectively; Fig. 5C). Importantly, the amount of activity associated with each deletion construct is comparable to that associated with Hap 4, which harbors the rs17539452 minor allele (Fig. 5C). These data indicate that the CREB and SOX10 consensus sequences are important for the observed enhancer activity of SH3TC2-MCS5.
SOX10 and CREB activate SH3TC2-MCS5 in Schwann cells
Our computational and functional data suggest that SH3TC2-MCS5 is responsive to the transcription factors SOX10 and CREB. To test this, we co-transfected the construct harboring SH3TC2-MCS5 Hap 1 upstream of a luciferase reporter gene with constructs to express wild-type SOX10 or CREB into SOX10-negative motor neurons (MN-1 cells) and SOX10-positive Schwann cells (S16 cells). In MN-1 cells, SOX10 and CREB both induced a >10-fold increase in activity compared with the untreated SH3TC2-MCS5 construct (P-values 1.92 × 10−7 and 1.83 × −10, respectively; Fig. 6A); however, an additive effect was not observed (data not shown). In S16 cells, SH3TC2-MCS5 Hap 1 enhancer activity increased by 62% (P-value 0.00013) and 130% (P-value 1.34 × −6) in the presence of additional SOX10 or CREB, respectively (Fig. 6B). Furthermore, an additive effect was observed when SOX10 and CREB were overexpressed together resulting in a 310% increase (P-value 9.06 × 10−13) in activity (Fig. 6B). These data indicate that SOX10 and CREB activate SH3TC2-MCS5 in a synergistic fashion in Schwann cells.
To determine if endogenous SOX10 and CREB activate SH3TC2-MCS5 in S16 Schwann cells, we performed luciferase assays in the presence of a dominant-negative (E189X) SOX10 expression construct (30) or ascorbic acid (33) to inhibit the activity of endogenous SOX10 and CREB, respectively. Expression of dominant-negative SOX10 reduced the enhancer activity of SH3TC2-MCS5 Hap 1 by ∼65% (P-value 4.25 × −10; Fig. 6C) and application of ascorbic acid reduced the activity of SH3TC2-MCS5 Hap 1 by ∼21% (P-value = 0.00078; Fig. 6D). To verify the effect of ascorbic acid on cultured S16 cells, we measured cAMP levels in untreated cells and those treated with 600 or 1000 μm ascorbic acid. This revealed a dose-dependent decrease in cAMP levels (Supplementary Material, Fig. S4A) consistent with the effect of ascorbic acid on the activity of SH3TC2-MCS5. Furthermore, to determine the specificity of ascorbic acid and dominant-negative SOX10 on SH3TC2-MCS5 activity, we performed luciferase assays on S16 cells transfected with the ΔCREBΔSOX10 SH3TC2-MCS5 luciferase expression construct in the presence of ascorbic acid or E189X SOX10. Neither of these conditions reduced luciferase activity (Supplementary Material, Fig. S4B and C), indicating that the effect of each requires the respective transcription factorbinding site. Collectively, these data are consistent with SOX10 and CREB regulating the activity of SH3TC2-MCS5.
CREB binds to SH3TC2-MCS5
The reduced activity of SH3TC2-MCS5 Hap 4 may be due to decreased binding of the rs17539452 minor allele to nuclear proteins in Schwann cells. To test this, we utilized electromobility shift assays (EMSAs). Briefly, a biotinylated-DNA probe containing the CREB and SOX10 consensus sequences and the major allele of rs17539452 ('Maj' in Fig. 7A) was incubated with nuclear protein isolated from S16 Schwann cells. In the absence of nuclear protein, unbound DNA probe migrates to the bottom of the gel (lane 1, Fig. 7A). When S16 cell nuclear protein is present, a distinct pattern of shifted bands is observed indicating that the probe binds to nuclear protein from Schwann cells (lane 2, Fig. 7A). Competition with increasing amounts of a cold 'Maj' competitor probe (lanes 3 and 4, Fig. 7A) caused a decrease in signal for one specific protein-bound DNA band (arrowhead, Fig. 7A). We infer that this band corresponds to a specific interaction between the 'Maj' biotin probe and S16 nuclear proteins. Competition with a probe designed around the minor allele of rs17539452 ('Min' in Fig. 7A) does not outcompete the binding as effectively as the 'Maj' competitor probe at 100× (compare lanes 4 and 5, Fig. 7A), consistent with the minor allele of rs17539452 having reduced binding to Schwann cell nuclear proteins.
To determine if the 'Maj' biotin-labeled probe binds specifically to CREB, we performed a 'supershift' EMSA using an anti-CREB antibody. The addition of increasing amounts of antibody resulted in a corresponding decrease in signal intensity of the same band outcompeted by the 'Maj' competitor probe in standard EMSAs (arrowhead in Fig. 7B, compare lane 2 to lanes 3 through 5). This suggests that CREB binds to the 'Maj' biotin probe and that the presence of the CREB antibody prevents this interaction, possibly by disrupting the DNA binding capabilities of CREB. These results are similar to previously published 'supershift' EMSA data using the same antibody employed here (34). Furthermore, CREB binding profiles generated by ChIP-Seq (35) show significant binding of CREB at SH3TC2-MCS5 in multiple human cell lines (Supplementary Material, Fig. S1B). Combined, these data indicate that CREB binds to the region surrounding rs17539452 and that the minor allele of this SNP impairs CREB.
Forskolin increases the expression of Sh3tc2 in Schwann cells in vivo
Our data thus far raise the possibility that SH3TC2 is a CREB target gene. To test this, we utilized forskolin, a well-characterized adenylyl cyclase activator that increases the levels of cAMP and activates CREB via the PKA pathway (36). Briefly, rat primary Schwann cells were treated with 3 μm forskolin for 24 h after which RNA was extracted and mRNA expression measured by quantitative RT-PCR. Treatment with forskolin induced a significant increase in the expression of Sh3tc2, Pmp22 and Egr2 (Fig. 7C); the latter gene encodes a master transcription factor of myelination. Importantly, the induction of Sh3tc2 is comparable to that of Pmp22, which harbors a characterized CREB response element in the promoter (37). Another Schwann cell gene, Sox10, whose expression is unaffected by altered cAMP production (38,39) is shown as a control. We also examined the effect of forskolin on the two loci flanking Sh3tc2: Ablim3 and Adrb2. These studies revealed that Ablim3 is not induced by forskolin (Fig. 7C) and that Adrb2 is not expressed in rat primary Schwann cells (data not shown). Combined, these data are consistent with CREB directly activating Sh3tc2 expression in Schwann cells via SH3TC2-MCS5.
Common SNPs at the SH3TC2 locus are associated with CMT disease severity
The identification of a regulatory SNP at SH3TC2 suggests that this variant (or other functional variants at SH3TC2) may modify CMT disease phenotypes. We studied a cohort of 407 exceptionally phenotyped patients with CMT1A that were collected under strict clinical protocols by the Inherited Neuropathy Consortium (INC). CMT1A is caused by a duplication event of a canonical area of chromosome 17 containing the PMP22 gene (17–19). On the background of this uniform mutation, we sought to identify genotypes at SH3TC2 that are associated with variable expression of key clinical outcomes in patients with CMT1A. Genome-wide SNP genotyping was utilized to test the hypothesis that common variation at the SH3TC2 locus (Supplementary Material, Fig. S4), particularly in the region surrounding rs17539452, is associated with CMT1A disease expression. Association was tested using the CAPL software (40). Nominal association was found with a 'difficulty walking' subphenotype (rs2082383, P-value = 0.048, 33 kb proximal to rs17539452; and rs6580586, P-value = 0.0194, 50 kb proximal), a 'decrease in feeling' subphenotype (rs1029942, P-value = 0.0141, 17 kb distal; and rs2082383, P-value = 0.00857, 33 kb proximal) and a 'foot dorsiflexion' subphenotype (rs4555784, P-value = 0.0479, 50 kb distal). There was no association with 'difficulty with balance' among the SNPs within 50 kb of rs17539452.
In addition to the region surrounding rs17539452, we examined SNPs within and adjacent to the SH3TC2 transcriptional unit (Supplementary Material, Fig. S4) for association with CMT1A disease severity. Three SNPs were associated with 'foot dorsiflexion' and 'decrease in feeling': (i) a nonsynonymous change (rs6875902, p.Ala468Ser; P-value = 0.0068), (ii) a 3′-UTR change (rs3763022; P-value = 0.0025) and (iii) an intronic change (rs2304034; P-value = 0.037). The non-synonymous change is moderately conserved with a GERP score of 0.802. Thus, our genetic analyses have revealed an association between genotypes at SH3TC2 and phenotypic outcome in CMT1A disease. However, due to the low minorallele frequency of rs17539452 (0.021 in individuals of European descent from the HapMap dataset), reliable association tests and linkage disequilibrium patterns could not be generated in our moderately sized patient cohort.
In this study, we describe transcriptional regulatory elements at the SH3TC2 locus, which is implicated in autosomal recessive CMT disease type 4C (CMT4C). Our analyses identified a promoter and downstream enhancer that are active in Schwann cells in vitro. The promoter region is regulated by the neural crest/Schwann cell transcription factor SOX10 based on multiple lines of evidence. Overexpression of wild-type SOX10 causes increased induction in promoter activity in SOX10-negative cells, and overexpression of dominant-negative SOX10, which prevents endogenous SOX10 from binding to DNA (30), causes decreased promoter activity in cultured Schwann cells. SOX10 regulation of the SH3TC2 promoter likely occurs via a head-to-head SOX10binding site located 96 bp upstream of the TSS, because mutating this site causes a dramatic reduction in promoter activity. Recent ChIP-Seq analysis of transcription factor binding sites in rat Schwann cells (28) revealed a single SOX10binding peak at the SH3TC2 promoter near the head-to-head SOX10 site described here. Since no other SOX10 consensus sequence within the promoter significantly contributes to regulatory activity, we conclude that our analyses have revealed the specific sequences required for SOX10 induction of the SH3TC2 promoter.
We also characterized a distal enhancer at SH3TC2 ∼150 kb downstream of the TSS. Based on similar experiments performed at the SH3TC2 promoter, we conclude that the downstream enhancer is regulated by the transcription factors SOX10 and CREB. Interestingly, while we present strong evidence of CREB binding at SH3TC2-MCS5, significant levels of SOX10 binding were not detected at this region in ChIP-Seq analyses performed on rat sciatic nerves (28). One possible explanation for this discrepancy is that this enhancer may be active at an earlier developmental stage than that assessed in the ChIP-Seq studies (see below). Indeed, based on the effect of deleting the SOX10binding site and the specificity of overexpressing wild-type and dominant-negative SOX10, we are confident that SH3TC2-MCS5 is a CREB/SOX10 response element. One of the difficulties of analyzing cis-acting transcriptional regulatory elements is determining the specific gene(s) that they regulate. This is especially problematic when a regulatory element maps to an intergenic region, as is the case with the downstream enhancer described here. However, we showed that SOX10 and CREB synergistically activate the downstream enhancer and that forskolin treatment, which activates CREB, induces Sh3tc2 mRNA transcription in vivo. In contrast, Adrb2 (the next locus downstream of Sh3tc2) is not expressed in Schwann cells and Ablim3 (the next locus upstream of Sh3tc2) is not induced by forskolin. Finally, the induction of Sh3tc2 expression by forskolin is comparable to that of Pmp22, which is a known CREB target gene in Schwann cells (37). Collectively, these data support the conclusion that SH3TC2 is regulated by SOX10 and CREB via the downstream enhancer.
SOX10 and cAMP have important roles in Schwann cell development and peripheral nervous system (PNS) myelination. SOX10 is expressed early in neural crest development and remains active in myelinating Schwann cells throughout development and adulthood (41–43). SOX10 interacts with other transcription factors important for regulating Schwann cell myelination including EGR2, POU3F2 and POU3F1/SCIP/OCT6 (26,44). While consensus sequences for EGR2 and POU3F2 were found within the SH3TC2 promoter (Fig. 2), these were not necessary for regulatory activity (Fig. 3). Furthermore, ChIP-Seq and expression studies have shown that SOX10 regulates Sh3tc2 independent of EGR2 (28). Intracellular cAMP plays an essential role in transitioning immature Schwann cells to myelinating Schwann cells by activating CREB (38,39,45,46). As mentioned above, co-expression of SOX10 and CREB revealed a synergistic effect on the activity of the downstream enhancer (SH3TC2-MCS5). Synergism between CREB and SOX10 was previously observed at the MITF locus in melanocytes (32), and other myelin genes are regulated by SOX10 and CREB including PMP22 (37,47,48), EGR2 (49–51) and OCT6 (38,52). While synergism between SOX10 and CREB has not been demonstrated at these latter three loci, it is clear that co-regulation by SOX10 and CREB is an important aspect of Schwann cell myelination.
Transcriptional regulation of SH3TC2 by cAMP/CREB may provide insight into how an increase in intracellular cAMP induces Schwann cell myelination (45). It was recently demonstrated that SH3TC2 interacts with the receptor ERBB2 in Schwann cells, resulting in internalization of ERBB2 from the adaxonal membrane (11). The Nrg1/ERBB signaling pathway is important for the regulation of Schwann cell proliferation and migration as well as for Schwann cell differentiation and myelination (10). The mechanism by which Nrg1/ERBB signaling switches between the two pathways is unclear; however, increasing intracellular cAMP causes the Nrg1/ERBB signaling cascade to direct Schwann cells to differentiate and myelinate the associated axon (45). Furthermore, impaired cAMP production results in the absence of myelinated peripheral nerve axons in Gpr126 mutant mice and zebrafish (38,39), and Sh3tc2-null mice exhibit hypomyelinated peripheral nerves (6). Combined, these data suggest that decreased cAMP production and impaired SH3TC2 function may disrupt a common pathway in PNS myelination. Our data indicate that cAMP/CREB induces SH3TC2 transcription. Therefore, we hypothesize that increased cAMP levels induce myelination by enhancing SH3TC2 transcription (likely via the downstream enhancer) and increasing ERBB2 internalization.
Characterization of the SH3TC2 promoter and downstream enhancer will facilitate the identification of pathogenic regulatory mutations that confer disease by disrupting SH3TC2 transcription. CMT4C is an autosomal recessive disease caused by loss-of-function mutations (7–9). Mutations disrupting a regulatory element may cause a loss-of-function effect by reducing or ablating SH3TC2 transcription. Indeed, while regulatory mutations have not been identified at SH3TC2, they may account for the missing heritability observed in patients with a CMT4C phenotype that are heterozygous for only one SH3TC2 coding mutation (15,16); however, our analysis of six such patients revealed no variants in the promoter or SH3TC2-MCS5 (data not shown). Additionally, the rs17539452 minor allele might directly cause disease in patients with CMT4C that are compound heterozygous for the minor allele and a second mutation—the predicted reduction in SH3TC2 expression caused by the minor allele, in combination with a second mutant allele at SH3TC2, may be enough to cause disease symptoms. Thus, we strongly recommend: (i) the inclusion of the regulatory elements described here in future mutation screens of patients with a recessive demyelinating CMT disease phenotype and (ii) evaluation of rs17539452 in CMT disease onset in patients with unexplained CMT4C disease.
An important, yet unexplored area of CMT disease research is the identification of genetic modifiers to explain the high clinical variability observed among patients with CMT disease, especially among those carrying the same genetic lesion (53). Determining the transcriptional regulation of CMT-associated genes will facilitate the identification of genetic modifiers that reside in regulatory elements and alter the expression of key PNS genes. We hypothesized that the SNP rs17539452 is an excellent candidate modifier of CMT disease since the presence of the minor allele in the downstream SH3TC2 enhancer perturbs CREB binding and reduces enhancer activity. CMT1A is the most common form of CMT disease and is caused by duplication of ∼1.5 Mb on chromosome 17, which harbors the PMP22 gene (17–19). While nearly all patients with CMT1A carry an identical duplication (54), it has long been noted that phenotypic expression varies even within the same family. On the background of this common PMP22 mutation, we tested if SNPs at SH3TC2 are associated with specific phenotypic outcomes in the largest cohort of uniformly phenotyped patients with CMT1A collected to date. Indeed, while our genetic studies did not have the power to detect an effect of the non-coding regulatory SNP described here (rs17539452), we did observe a number of significant correlations (P < 0.05) between SNP genotypes and CMT1A disease severity. These data are hypothesis driven due to the known importance of SH3TC2 in PNS myelination, however replication in an additional patient cohort will be required to validate these findings. Additionally, it will be important to demonstrate a correlation between the genotype of the SNPs studied here and SH3TC2 expression or protein function; however, the former studies are complicated by the Schwann cell-specific expression of this gene.
Finally, the data presented here may have implications for designing strategies to treat patients with CMT1A disease. Increased expression of PMP22 is the pathogenic mechanism of CMT1A disease, and it has been proposed that decreasing PMP22 expression will alleviate the clinical phenotype. Upon finding that CREB positively regulates the PMP22 promoter (37), it was posited that treatment with ascorbic acid, which reduces CREB function, may reduce PMP22 expression and benefit patients with CMT1A. Indeed, mice with a PMP22-overexpression-induced neuropathy showed dramatic behavioral and histopathological improvement upon treatment with ascorbic acid (55). However, initial clinical trials on young and adult patients with CMT1A have not revealed any benefits of ascorbic acid treatment (56–59). One reason for the lack of improvement in patients may be due to a deleterious effect of ascorbic acid on CREB response loci such as EGR2, OCT6 and SH3TC2, as well as other, undiscovered CREB target loci. Furthermore, the success of ascorbic acid treatment in mouse may reflect differential effects on gene expression between rodents and human. Interestingly, the CREB-responsive major allele of the SNP we characterized in the downstream enhancer (SH3TC2-MCS5) is human specific. These data also raise the possibility that certain human loci have developed a special requirement for CREB function in Schwann cells. Addressing this will require defining the full panel of CREB target genes in Schwann cells and assessing for differences in gene expression and regulatory activity between human and rodents.
In summary, we have characterized transcriptional regulatory elements at SH3TC2 that are activated by the transcription factors SOX10 and CREB. These findings revealed: (i) additional regions for mutation screening in patients with CMT4C, (ii) a SNP allele that dramatically reduces the activity of a downstream enhancer, (iii) a novel target gene of CREB in Schwann cells and (iv) specific SNPs at SH3TC2 that are associated with phenotypic differences in patients with CMT1A. As research on the genetics and genomics of CMT disease moves further toward whole-genome interrogation, efforts should focus on a broader assessment of regulatory SNPs in peripheral nerve function and CMT disease etiology, which will likely reveal pathways to exploit for therapeutic development.
Comparative sequence analysis at the SH3TC2 locus
The following sequences from orthologous SH3TC2 loci were obtained from the UCSC Genome Browser (60): human (chr5: 148 188 390–148 501 247, hg18), mouse (chr18:62 047 106–62 352 470, mm9), rat (chr18:57 898 589–58 181 925, rn4), dog (chr4:62 992 595–63 262 602, canFam2), cow (chr7:62 226 792–62 510 218, bosTau6) and opossum (chr1:377 577 553–377 972 236, monDom5). Repetitive DNA sequences were masked using the UCSC Genome Browser 'Sequence Formatting Options' and each genomic sequence was submitted to the MultiPipMaker alignment algorithm (21). The human genomic sequence at SH3TC2 was used as the reference, and the 'Search both strands' and 'Chaining' options were employed. Subsequently, the MultiPipMaker 'acgt' alignment file was submitted to the EP algorithm (http://research.nhgri.nih.gov/exactplus/) (22) using the following settings: minimum length of exact match to seed = 5, minimum number of species to seed = 6 and minimum number of species to extend an alignment = 6.
Transcription factorbinding site predictions
To identify Schwann-cell relevant transcription factorbinding sites in the SH3TC2 promoter and SH3TC2-MCS5, we submitted each genomic sequence (Table 1) to the TRANSFAC transcription factor prediction program using the MATCH algorithm (25), the 'minimize the sum of both error rates' setting, and the following matrices: V$BRN2_01, V$EGR2_01, V$POU3F2_01, V$POU3F2_02, V$SOX10_Q6 and an in-house SOX10 consensus sequence 'ACAMD' (where 'M' is a C or A nucleotide and 'D' is a G, T or A nucleotide). To identify transcription factor consensus sequences that are differentially predicted on the major and minor alleles of rs17539452, we submitted 24 bp of genomic sequence surrounding each allele [GGATGACTACAcGTCACAGTGGCA (major allele) and GGATGACTACAtGTCACAGTGGCA (minor allele)] to the TRANSFAC transcription factor prediction program using the MATCH algorithm (25), the 'minimize the sum of both error rates' setting and the 'vertebrate, non-redundant' matrix database.
Expression vector construction
Oligonucleotide primers containing attB1 and attB2 Gateway cloning sequences (Invitrogen) were designed for PCR-based amplification of the SH3TC2 promoter and each SH3TC2-MCS region (Supplementary Material, Table S1). Subsequent to PCR amplification and purification, each genomic segment was cloned into the pDONR221 vector using BP Clonase (Invitrogen). Resulting constructs were genotyped by digestion with BsrGI (New England Biolabs) and subjected to DNA sequence analysis to ensure sequence specificity. Each resulting pDONR221 construct was recombined with a destination vector harboring a minimal promoter directing a luciferase reporter gene (pE1B-luciferase) (22) using LR Clonase (Invitrogen). Successful cloning of each genomic segment upstream of the luciferase reporter gene was assessed by genotyping with BsrGI (New England Biolabs).
Site-directed mutagenesis reactions were performed using the QuikChange II Mutagenesis Kit (Agilent Technologies) and appropriate mutation-bearing primers (Supplementary Material, Table S1). Mutagenesis primers were designed to delete or mutatespecific transcription factor consensus sequences or to introduce specific alleles of SNPs. Mutagenesis was performed in pDONR221 constructs (see above), and each resulting clone was subjected to DNA sequence analysis to ensure that only the desired mutation was introduced into the insert. Correctly mutagenized inserts were recombined into the luciferase reporter gene construct as described above.
Cell culture and luciferase assays
Immortalized rat Schwann cells (S16) (61) and mouse spinal motor neurons (MN-1) (62) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mml-glutamine. Cells were incubated at 37°C in 5% CO2. 1 × 104 cells were plated in a 96-well culture plate (Corning Life Sciences) and incubated overnight. Subsequently, cells were transfected with luciferase expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications. Each well was transfected with 200 ng of the indicated luciferase expression construct and 2 ng of a renilla expression construct (to control for transfection efficiency and cell viability) in 50 µl of Opti-MEM and 0.25 µl Lipofectamine (Invitrogen) cocktail as previously described (63). For overexpression of each transcription factor, 100 ng of wild-type SOX10, dominant-negative E189X SOX10 (30) or wild-type CREB (Clontech) expression constructs were co-transfected into each reaction. Cells were incubated in the transfection solution for 4 h and then cultured in normal growth media. For ascorbic acid treatment, growth media was supplemented with 600 µm ascorbic acid (Sigma–Aldrich) and the effect on cAMP levels was evaluated using the cAMP-Glo™ Max Assay (Promega) according to the manufacturer's specifications. After 48 h, cells were lysed in 20 µl of passive lysis buffer (Promega) for 1h and 10 µl of each lysate was transferred to a polystyrene 96-well assay plate (Corning Life Sciences). Luciferase and renilla activities were then determined using the Dual Luciferase Reporter Assay System (Promega) and a Glomax Multi-Detection System (Promega). Each experiment was performed at least 24 times using at least two different DNA preparations per construct. The ratio of luciferase to renilla activity and the fold increase in the ratio relative to a control luciferase expression vector with no insert ('Empty' in figures) were calculated. The mean (bar height in figures) and standard deviation (error bars in figures) were determined using standard calculations. Statistical significance was performed using a two-tailed Student's t-test where * and ** indicate a P-value <0.01 and 0.001, respectively.
5′ Rapid amplification of cDNA ends
First-strand cDNA was generated from RNA isolated from rat cultured Schwann (S16) cells using an oligonucleotide primer designed within exon 4 of the rat Sh3tc2 gene (5′-tttcctccagggtcttgaagg-3′). The cDNA sample was subsequently TdT-tailed using the 5′ RACE System (Invitrogen) and sequential PCR reactions were performed using nested primers (Supplementary Material, Table S1). PCR products were separated on 1% low-melt agarose gels, excised and purified using the QIAquick Gel Extraction Kit (Qiagen), and subjected to DNA sequence analysis.
Electromobility shift assay
S16 nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Fisher Scientific). 5′-biotin-labeled and unlabeled complimentary oligonucleotides (Supplementary Material, Table S1) were annealed to form biotinylated and competitor probes, respectively. EMSAs were performed using the LightShift Chemilumincescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer's specifications. Briefly, 5 μg of S16 nuclear extract was incubated with or without an unlabeled competitor probe (10× and 100×) in a 20 μl binding reaction containing 1× binding buffer and 50 ng Poly(dI.dC) for 20 min at room temperature. Subsequently, 20 fmol of biotinylated probe was added to each binding reaction and incubated for an additional 20 min at room temperature. Binding reactions were electrophoresed on a 6% TBE DNA retardation gel (Invitrogen) and transferred to a positively charged nylon membrane (Roche) using a semi-dry transfer unit (Bio-Rad). DNA was crosslinked to the membrane using the Spectrolinker 2000 (Spectronics). Chemiluminescent detection of the biotinylated probes was performed with a streptavidin-horseradish peroxidase conjugate (Thermo Fisher Scientific) and exposed to X-ray film. For 'supershift' EMSAs, varying amounts (0, 3, 4 and 5 μg) of CREB antibody (C-21; Santa Cruz Biotechnology) were incubated with S16 nuclear extract prior to adding the labeled probe.
Western blot analyses
Cultured S16, MN-1 and HeLa cells were harvested and resuspended in 37.5 μl of lysis solution containing 10 mm Tris–HCl/1 mm EDTA/50 mm NaCl (Invitrogen) and 12.5 μl of loading buffer containing SDS (Invitrogen). After incubation at 95°C for 5 min, the protein lysate was electrophoresed on 4–12% Bis–Tris gels (Invitrogen) at 125 V for 100 min in running buffer containing SDS (Invitrogen). Samples were then electroblotted onto nitrocellulose membranes at 25 V for 100 min in NuPAGE transfer buffer (Invitrogen). Membranes were blocked in 1× PBS/0.05% Tween 20/5% non-fat dry milk for 18 h at 4°C and then incubated with a 1:1000 dilution of anti-CREB antibody (C-21; Santa Cruz Biotechnology) in blocking solution for 1 h at room temperature. Membranes were washed three times in 1× PBS plus 0.05% Tween 20, incubated with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature, washed three times as described above and incubated with DAB substrate (Roche Diagnostics), according to the manufacturer's instructions.
Sh3tc2 expression studies
Rat primary Schwann cells were cultured as previously described (64). Cells were treated with 3 μm forskolin (Sigma–Aldrich) in DMSO or an equivalent volume of only DMSO (control) for 24 h after which total mRNA was extracted using Trizol (Invitrogen). One microgram of total RNA from each sample was used to prepare cDNA as previously described (65). Quantitative RT–PCR was performed by monitoring in real time the increase in fluorescence of the SYBR-GREEN dye as described (66) using the TaqMan 7000 Sequence Detection System (Applied Biosystems). Relative amounts of each transcript between samples were determined using the comparative Ct method (67) and normalized to levels of 18S rRNA. Primer sequences are listed in Supplementary Material, Table S1.
Genotyping DNA samples from patients with CMT1A
Four hundred and seven patients with the classic CMT1A-causing PMP22 duplication were ascertained under a protocol developed by the INC. IRB approval was obtained from all participating sites and all patients were consented accordingly. Detailed phenotypic assessment was performed by board-certified neurologists and, for this study, four categories of outcome were defined that capture the major features of peripheral neuropathies: 'difficulty walking', 'decrease in feeling', 'difficulty in balance' and 'foot dorsiflexion'. Genotyping was performed using the Illumina Human OmniExpress 12v1; however, only SNPs in the vicinity of SH3TC2 were extracted for this study (Supplementary Material, Fig. S4). Association was tested using the Combined Association in the Presence of Linkage (CAPL) software (40). CAPL performs statistical association tests, allowing for the inclusion of family data and population substructure.
Supplementary Material is available at HMG online.
This work was funded by grants from the National Institute of Neurological Diseases and Stroke to A.A. (NS073748) and J.S. (NS075269) and the Rare Disease Clinical Research Center for Inherited Neuropathies (U54NS065712). M.H.B. was a recipient of the Sir Keith Murdoch Fellowship from the American Australian Association, and F.B. was supported by the Princess Beatrix Spier Fund (PBF; grant WAR08-18).
The authors thank Chani Hodonsky for excellent technical assistance; Richard Quarles for the S16 cell line; Kurt Fischbeck for the MN-1 cell line; Jim Lupski, Ken Inoue and Michael Wegner for SOX10 expression constructs; the laboratory of Miriam Meisler for thoughtful discussions; Pavel Seeman and Kurt Fischbeck for DNA samples from patients with CMT4C; and the members of the Inherited Neuropathy Consortium for contributing DNA samples from phenotyped patients with CMT1A, which were essential for the association studies presented here.
Conflict of Interest statement. None declared.


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David Byrne's JOAN OF ARC: INTO THE FIRE Extends Again at The Public Theater
by BWW News Desk - Feb 21, 2017
The Public Theater announced today a two-week extension for the world premiere musical Joan of Arc: Into the Fire. (more...)
Community Spirit and a Great Show at the WhatsOnStage Awards
by Debbie Gilpin - Feb 20, 2017
Awards season is in full swing now, so this weekend was the turn of the 17th Annual WhatsOnStage Awards. Held at the Prince of Wales Theatre, it prides itself on being the only event where winners are voted for exclusively by theatregoers. (more...)
BWW Interview: Jordan Craig of THE PHANTOM OF THE OPERA
by Justin Cole Adams - Feb 20, 2017
Jordan Craig talks about how Atlanta theatre prepared him for the PHANTOM national tour. (more...)
Lea Salonga Headed Down Under with Tunes from MISS SAIGON, HAMILTON and More
by BWW News Desk - Feb 2, 2017
Broadway superstar and Disney legend, Lea Salonga, will be kicking off a series of five concerts in Australia beginning with three sold out shows at the iconic Sydney Opera House with the Sydney Symphony Orchestra, followed by two shows at Melbourne's Hamer Hall with Orchestra Victoria. (more...)
BWW Morning Brief February 17th, 2017 - COME FROM AWAY, CRAZY FOR YOU and More!
by Jessica Khan - Feb 17, 2017
Good morning, BroadwayWorld! This weekend's big news: COME FROM AWAY and CRAZY FOR YOU open on Sunday! (more...)
Caroline Bowman, Erin Dilly, Alton Fitzgerald White and More Set for BROADWAY SPOTLIGHT at Harbor Lights Theater Company
by BWW News Desk - Feb 16, 2017
A special event entitled Broadway Spotlight, March 18, at 8:00 p.m. at the College of Staten Island's Williamson Theater benefits The Harbor Lights Theater Company. (more...)
BWW Morning Brief February 16th, 2017 - Broderick Back Onstage, THE PRICE and More!
by Jessica Khan - Feb 16, 2017
Good morning, BroadwayWorld! Today's big news: Matthew Broderick returns to the stage and THE PRICE begins on Broadway! (more...)
BWW Preview: Full Circle Productions to Present BRINGING BACK BROADWAY - A Musical Review, 3/2-7
by Noelle Hannibal - Feb 16, 2017
A cast of 27 local Montreal performers, ages 8-70, will present BRINGING BACK BROADWAY, A Musical Review from March 2-7, 2017 at Congregation Beth Tikvah,136 Westpark Blvd, Dollard des Ormeaux, QC. The two hour long Broadway musical revue will highlight songs from ANNIE, WICKED, ALADDIN, THE LITTLE MERMAID, Miss Saigon, FIDDLER ON THE ROOF, CHESS, WAITRESS, HAMILTON, THE SOUND OF MUSICA and LES MISERABLES. (more...)
Photo Coverage: Get Up Close with the Cast of MISS SAIGON!
by Jennifer Broski - Feb 16, 2017
Rehearsals are underway for Cameron Mackintosh's acclaimed new production of Boublil and Schonberg's legendary musical Miss Saigon, which will return to its original home, The Broadway Theater (1681 Broadway) beginning performances Wednesday, March 1. The original production of Miss Saigon opened at the same theater on April 11, 1991 and played there through January 28, 2001. (more...)
WONDERLAND Gets a Rock & Roll Makeover at ATC Kids This March
by BroadwayWorld JR - Feb 16, 2017
The Atlantic for Kids 2016/2017 Season will resume Saturday, March 4 with the family rock concert meets live theater adventure Wonderland: Alice's Rock & Roll Adventure, a New York premiere musical based on Lewis Carroll's beloved Alice and Wonderland tales, featuring book and lyrics by Rachel Rockwell, music and lyrics by Michael Mahler, and direction by Marshall Pailet. (more...)
BWW TV: The Heat Is Back On! Meet the Company of MISS SAIGON
by TV - Press Previews - Feb 15, 2017
Rehearsals are underway for Cameron Mackintosh's acclaimed new production of Boublil and Schonberg's legendary musical Miss Saigon, which will return to its original home, The Broadway Theater (1681 Broadway) beginning performances Wednesday, March 1. The original production of Miss Saigon opened at the same theater on April 11, 1991 and played there through January 28, 2001. (more...)
Photo Flash: Go Inside Rehearsals for MISS SAIGON's Broadway Return!
by BWW News Desk - Feb 15, 2017
Rehearsals are underway for Cameron Mackintosh's acclaimed new production of Boublil and Schonberg's legendary musical Miss Saigon, which will return to its original home, The Broadway Theater (1681 Broadway) beginning performances Wednesday, March 1. The original production of Miss Saigon opened at the same theater on April 11, 1991 and played there through January 28, 2001. (more...)
The Musical Stage Company Celebrates New Name; FUN HOME Canadian Premiere Headlines 2017-18 Season!
by BWW News Desk - Feb 14, 2017
With great pride and excitement today, Acting Up Stage Company makes a series of major announcements about a significant expansion, along with a robust 14th season, new musical development initiatives, major producing partnerships, and a brand new name and vision. (more...)
BWW Interviews: Juan Pablo Di Pace en 54 Below
by Juan-Jose Gonzalez - Feb 14, 2017
Hablamos con Juan Pablo Di Pace acerca de su show en 54 Below el próximo 21 y 22 de febrero, de su éxito en Netflix con FULLER HOUSE y de este nuevo capítulo de su carrera. (more...)
BWW Interview: Juan Pablo Di Pace Gets Ready for His 54 Below Debut
by Juan-Jose Gonzalez - Feb 14, 2017
On February 21st and 22nd, TV star Juan Pablo Di Pace will take the stage at New York's 54 Below with a his new show, highlighting his song and dance skills, so far seen in London and Madrid. (more...)
SEASONS OF LOVE Concert Featuring Cast from HAMILTON, MISS SAIGON, A BRONX TALE, and More Tonight
by BWW News Desk - Feb 12, 2017
Tonight, Lexi Lawson, currently playing 'Eliza Schuyler' in Broadway's Hamilton, will join starry alumni of the Broadway, National Tour, and Off-Broadway companies of the Pulitzer Prize winning musical RENT. The special Valentine's Day tribute to the hit musical will take place on February 12 at The Cutting Room. The show will include songs from the musical, as well as special, unheard stories from former cast members, includingRichard H. Blake (A Bronx Tale, Legally Blonde), Antoine L. Smith (Miss Saigon), Corey Mach (Godspell, Hands on a Hardbody), and Christina Sajous (American Idiot, Spider-Man: Turn Off the Dark). (more...)
Top Stories You Missed on BWW This Weekend - 2/11-2/12/2017
by - Feb 12, 2017
Hottest Articles on BroadwayWorld.com from this weekend Sunday, February 12, 2017 - Sunday, February 12, 2017. (more...)
Photo Flash: Passionate Play 33 VARIATIONS Takes Off in LA
by BWW News Desk - Feb 11, 2017
Actors Co-op (Ovation Award-Winner 2014 Best Intimate Theatre Musical for 110 in the Shade) presents the third show in its celebratory 25th Silver Anniversary season with the Los Angeles intimate theatre premiere of 33 VARIATIONS by Moisés Kaufman, choreography by Michelle Parrish and directed by Thomas James O'Leary (Next to Normal). 33 VARIATIONS will preview on Thursday, February 9 at 8pm and will open on Friday, February 10 at 8pm and run through Sunday, March 19 at the Actors Co-op David Schall Theatre, 1760 N. Gower St. (on the campus of the First Presbyterian Church of Hollywood) in Hollywood. (more...)
Photo Flash: Disney's LITTLE MERMAID Set to Dazzle at Broward Center in Fort Lauderdale
by BWW News Desk - Feb 11, 2017
A spectacular new production of Disney's The Little Mermaid kicks off at The Broward Center for the Performing Arts on Wednesday, February 22, 2017 for a limited two week run. Featuring the critically acclaimed work of Director Glenn Casale, this beloved tale of a young mermaid's coming-of-age adventure is told like never before. Previously announced casting includes Diana Huey as Ariel and Matthew Kacergis as Prince Eric, with Broadway's Jennifer Allen as Ursula and Steven Blanchard as King Triton. Melvin Abston, Connor Russell and Jamie Torcellini join the cast as Sebastian, Flounder and Scuttle. (more...)
STAGE TUBE: Watch Highlights of THE FULL MONTY at The Engeman Theater
by Stage Tube - Feb 10, 2017
The John W. Engeman Theater presents THE FULL MONTY, now running through March 5, 2017. (more...)
Sheridan Smith And Chris Peluso Star In FUNNY GIRL
by BWW News Desk - Feb 10, 2017
???????Sheridan Smith and Chris Peluso lead the cast of Funny Girl which comes to The Bristol Hippodrome from March 21st - 25th having enjoyed critically acclaimed and record-breaking sold-out runs at the Menier Chocolate Factory and Savoy Theatre. (more...)
Photo Flash: Brian Stokes Mitchell, Danny Burstein and More Celebrate Adrienne Arsht's 75th Birthday
by BWW News Desk - Feb 10, 2017
Philanthropist Adrienne Arsht (as in the Adrienne Arsht Center for the Performing Arts in Miami) hosted a glamorous 75th Birthday Party on Saturday night at the Plaza. Many Broadway and Metropolitan Opera performers, all of whom are Adrienne's close friends, performed for her as their birthday gift. Scroll down for photos! (more...)


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by Adam Plowden | 22nd September 2016
The SYRP Slingshot is a wirecam setup that adds dynamic motion and expansive length to time-lapses at an affordable cost. Have you ever wanted to capture longer motion timelapses where a slider track just isn't long enough? Enter SYRP Slingshot, a wirecam package that can reach distances of up to 300 feet (100m) with up to 3-axis camera movement using the SYRP Genie and Genie Mini. The SYRP Genie works as the motion control device, moving the carriage along parallel wires of your desired length, whether that's 25m or 100m. Inter-operable with your camera, the Genie moves the carriage with the time-lapse function to create smooth and unique perspectives that can't be achieved without the need to spend lots of money. Some of the time-lapse shots in their demo video are incredible! SYRP have designed the Slingshot with simplicity of setup and control in mind, with two mounting brackets, straps, wires, carriage and Genie all fitting into a compact bag. The maximum payload of 8kg is enough for a weighty camera and lens combo. The example video shows a Canon DSLR and lens weighing around 4kg which should give you an idea. It seems that as the Genie controls the intervalometer for movement, a continual move along the wires might not be possible. However, for time-lapse photography, it certainly goes above and beyond what you can normally capture with a static shot or even short distance movement. The SYRP Slingshot is available in a number of packages, with the standard 'Slingshot' package consisting of – 1 Slingshot (25m Slingshot rope and 25m Genie rope). 1 Slingshot carry bag That kit comes in at $989.00, a very affordable package compared to, for example, the Kessler Second Shooter. Of course, you'll need a SYRP Genie to actually move the carriage, so you can get a bundle with a Genie, the link cable and ball head for $1887. The Slingshot is available now! Are you looking to shoot expansive time-lapse scenes and need a setup like this? Let us know in the comments!Read more
by Richard Lackey | 11th April 2016
While EROVR sounds like something NASA might send to Mars, it's actually a very clever multipurpose cart that filmmakers may find very useful. EROVR is not necessarily aimed at the film industry, but it's a nifty concept with countless potential duties on set that are immediately apparent. Introducing EROVR Currently running a campaign on indiegogo, EROVR USA has developed a "multi-functional personal carrier that transforms into 10 configurations to suit any task at hand." According to the EROVR indiegogo page: Storage Mode Movers Dolly Half Cart with Handle Half Cart with Handle + Fence Panels Flat Cart No Handles Flat Cart One Handle Baggage Cart Wagon Hand Truck Hand Truck with Side Panels The EROVR cart can be ordered for $195 from the EROVR indiegogo page and there are various accessory packs as well. I can see this cart being incredibly useful for loading and unloading gear, and shifting from place to place on the grounds of large sprawling locations. I've been on countless shoots in and around large hotel and resort complexes where the one or two actual camera carts we had on hand could be found loaded up with all kinds of things other than just being used for camera prep. A few of these would have made a world of difference on many shoots, and when it comes to packing everything up, the cart collapses into a very tight package that can just about fit anywhere for transport and storage. Check out the indiegogo campaign page for the latest information and updates.Read more
by Tim Fok | 8th April 2016
Here's a nice little grip system that can save you time on set. The Ninja Dolly is versatile & compact, made in the U.S and benefits from the use of standard domestic tubing for the track. Primarily a dolly but also much more, place it on the ground for a high hat or rig it to a car with its four industry standard baby pin receivers. Introducing the Ninja Dolly I'm not sure what I see most of nowadays, Sony and their affordable 4K cameras or working DPs who have ventured into making an all-in-one grip product. The Ninja Dolly system (to the dismay of all Sony fanboys) falls into the latter category but shakes things up a little with its quick and easy switch between various guises. As a tracking dolly, the Ninja Dolly is a compact and universal system; a neat flat base, strong & thin with easy lift handles and a compact 75/100mm bowl adaptor in the middle for tripod heads. V-shaped wheels with flat tops ensure a wide variety of compatible track poles, as well as the option to track along a flat floor without any track. The Ninja Dolly ships with no track, just pop down your local hardware store and grab some standard scaff tubing in the 1 5/8″ region. The feet are the end blocks to your tracking system. These are adjustable in width according to your setup and payload and can either sit on the floor or attach to conventional grip heads via 1 or 2 baby receivers. 4 of the same industry standard baby receivers can be found on Ninja Dolly flat base. These protrude further than the wheels themselves meaning you can seamlessly switch between dolly and high hat configuration; they operate as the protection to the wheels when you place the Ninja Dolly on a flat surface too. However, their use doesn't stop there; you can use the Ninja Dolly as a platform for car mounting. Or stick some suction cups on the receivers for more tricky configurations. Owner and creator, Brent Buntyn, takes pride in the 2-year journey it's taken to get to this point—not only in production but also testing and refining the Ninja Dolly in-the-field as a working DP. I think this shows; it's a simple system that catches my eyes due to its ability to switch seamlessly through configurations. Package price for the Ninja Dolly with 2 Feet Rail Brackets (no rails) is $799+shipping.Read more
by Giorgio Sironi | 6th April 2016
This is a guest review by Italian cameraman Giorgio Sironi, based in Vienna, Austria, of the Shootools Slider Pro. As I started looking for a new slider, I didn't know what I wanted, but I did know what I didn't want. I have seen, used or tested many different sliders, big ones and small ones. Good ones and atrocious ones. I talked to many colleagues, and I was intrigued of the various requirements each of them had. A tool is not just a tool, but more what the person using it makes out of it. For good or for bad. So what were my necessities? Putting it simply, I wanted something light that I could travel with. I wanted something long on the tripod but even on the ground. I wanted something sturdy, if you can carry an Alexa, you can carry an A7. I wanted something affordable just to keep my wife at bay. And I wanted something aesthetically pleasing – just to fit the global image. After this ego trip, I began my search. A few sliders came to my mind, full aluminum, with retracting rails, cheaper ones, sturdier ones. After a long search, I saw an ad from Shootools. The product looked interesting and it being an Italian firm I decided to call them up. The first contact was friendly. The sales manager took a lot of time to explain to me the ups and downs of the slider. I told him my concerns, and he gave me real answers. So I decided to order it. The Shootools Slider Pro 100 ShooTools is an Italian manufacturer who has already produced items like the Slider Modula with extension rails, or the dolly 360 with motion control (something worthy of a look in the future). Their latest product is the slider pro (60, 80, 100 and 150). Let's take a look at its main features. This slider boasts a new Magnetic Field Technology. A magnetic field is generating between the lateral stoppers and the carriage while running close the end-stop and counteracts at the beginning/end of the track, by slowing down the run of the carriage while reaching the end-stop. That means that when I push the carriage toward the end of the rail a kind of invisible cushion warns me and stops me before hitting the end. Spooky but intriguing. You can even shoot the carriage toward the end, and it bounces back without hitting the end. No more hard hits. Its carriage as no less than eight high precision steel ball bearings for the maximal fluidity of movement and performance. But everybody knows that you can have the best bearings on crappy wheels, and the result is of course crap. Apparently not here, the Shootools trademark wheels are made of an innovative ST-66 technopolymer, they guarantee maximal smoothness and extreme fluidity, in temperatures ranging from the lowest -50 up to torrid 130 degrees Celsius. As a bonus, it allows high payloads without fear of deformations. A nice touch is the possibility to quickly adjust the friction and a nice, fast and very safe brake which allows a good blockage of the carriage and its load even if it is on the heavier side. It weights just 2,8 Kgs, and we are talking about 100 cm worth of slider. The shorter ones go down to around 2 kilos. Even with this featherweight, the slider feels stable and sturdy. Why that happens is best described by the guys from ShooTools: "the rail has got an absolute exclusive, unique layout, designed and extruded by ShooTools. Light and at the same time strong, the ShooTools Slider Pro has a peculiar trapezoidal section intended to have the maximum steadiness possible. Did we already mention that it can carry up to 50 Kgs? No? Well, it does. The carriage, the rails, and the structure are made for this kind of payload. Is your tripod ready for this too? It has two movable tripod fixing points. They are two base plates with two threaded holes each (3/8" and 1/4") that you can move on the whole length of the slider and position wherever you want. In addition to that, you have four small feet, with rubber inlays, that you can position where you want. It looks and feels good. The wooden end stops are one nice touch, they give the slider that fancy touch and offer a unique grip. But they are not the only design united to tech features, "Each component undergoes two distinct types of treatment: the first one, by shot peening through ceramic spheres, increases the hardness and the resistance to fatigue of the object and, also, provides a pleasant hi-grip finish. The second treatment, anodizing at 30 microns, protects the device from any external agent." That means that the finish is not just black and sleek but resistant and pleasant to the touch too. It has lifetime warranty; that makes my Italian friends not only very convinced of their product but even very optimistic about our working, social and economic future! An official product video demonstrating the moves: My Conclusion I didn't use the slider for too long, and I am not impressed by sleek and Black, but if I can give my 5 cents I would say that is an excellent piece of equipment! For now, it feels, looks and works as a professional tool. I can carry it easily, even on the tripod. I can take it down from the tripod, put it on the floor, on a table or top of a crate in just a few seconds, and I still have the whole 100 cm for my use. The carriage is very smooth, and it took every camera we threw at it. My Pros: Very light, to carry and to use.Plug and play, no adjustments no maintenance. Plug and play, no adjustments no maintenance. Nice new features, like the magnetic field or the honeycomb structure. It looks good. The optional bag is well made. It is made in Italy. I am Italian. Nice responsive and helpful Sales team. My Cons: The built-in feet are nice but not enough for stability; I suggest to buy the optional wider feet for more stability. If I put a tripod plate on the slider base plate I have no holes to put the optional wider feet, that means that every time that I want to use the feet, I have to unscrew the tripod plate and move the slider plate in the correct position. After a phone call with ShooTools, I ordered two extra slider plates and mounted them on the slider. This offers me four access points for feet, tripods or hanging possibilities. It adds around 200 grams to the whole weight, but it is worth it. ShooTools even assured me they are going to look into the issue and probably come up with extra smaller plates for the feet. The magnetic field is nice, but it eats a few centimeters away on both sides, that makes the slider a bit shorter than a full meter. All in all a nice product. In its price range, I think it is a good choice. Would I suggest it to a friend? Well, as I said at the beginning, just if it fits his necessities. But if it does, hell yeah! ———————————————————————————————————————— MAIN FEATURES Lightness-record Magnetic Field Technology Friction System Wheels slide inside the rail. Horizontal load ± 50 Kg Total absence of maintenance Integrated feet Ready for motorization Lifetime warranty IN THE BOX Camera Slider PRO Integrated feet Setup Keys Instruction Manual Non-woven fabric wrapper TECHNICAL DATA Weight: Camera Slider Pro 60: 2,20 kg Camera Slider Pro 80: 2,50 kg Camera Slider Pro 100: 2,80 kg Camera Slider Pro 150: 3,55 kg Load: ± 50 Kg Material: stainless steel, Anticorodal alloy, Technopolymer; end-stops: wood Treatments: Shot peening, Organic Anodization ————————————————————————————————————————Read more
by Nic Divischek | 5th October 2015
Dan Eckert from CamTrac wanted to design an all-purpose camera dolly and has turned to Kickstarter to make his invention available to the public. Introducing the Camtrac. Whether for Timelpase, Hyperlapse or regular video camera moves, this looks like a cool toy for filmmakers who want to go the extra mile and create stunning images. The Camtrac will run on almost any surface or rails. The motorized dolly has adjustable and removable components and wheels, thus making it highly functional and suitable for most set-up situations. Whether you want to clamp a tripod, mono-pod or hi-hat to it, or let it grip to any railing you can find, the Camtrac is a promising tool for all filmmakers to get that shot you always wanted to get, but didn't have the means to do so. The software is still in development to create accurate timelapses and hyperlapses, however it all seems to be on the right track. The Kickstarter project just started, so if you are interested in being one of the first to receive this great gear, head over to Camtrac's kickstarter page. They are still short of their goal a fair bit, but there are are still 23 days to go.Read more
by Sebastian Wöber | 20th September 2015
The Korean company Motionnine is fairly new to the equipment market, but has come up with quite a few inventive camera accessory ideas. One of the newest gadgets they showed off at IBC 2015 is an affordable compact slider that we liked. The new compact slider goes by the name Motionnine Single Linecam and is available with a length of 60 cm and 80 cm. The slider has a break and simplistic dampening system that worked quite well when we tried it at the booth and will be available in October for $185. Find out more on the Motionnine website.Read more
by Tim Fok | 21st August 2015
LOKI is a pocket-sized multi-purpose rig that transforms into a Shoulder Rig, Cage, Underslung Rig and Dolly. UK based creators Scratch Ideas are hosting a Kickstarter campaign to get the project off the ground. The base unit of LOKI folds down to a size similar to that of a DSLR battery grip; this is grounds to make up the majority of the LOKI shoulder rig & underslung rig and with a couple more components you can build the cage and 4-wheeled dolly. LOKI as a shoulder rig has a 20kg weight limit, and all joints are made from aluminium rosettes for ease of adjustment: "We included serrated steel locking plates on all the joints to offer loads of articulation, but more importantly, to ensure that once your rig is set-up, it will not move or slip. " The shoulder rig module offers two 15mm rods for standard accessories and the cage offers an array of additional 1/4 20″ threads for further mounting. The dolly configuration requires two wheeled axles and takes advantage of LOKIs quick release rosette system. Here's a look at all the available components and pricing for the LOKI: Personally, I've never been a huge fan of an all-in-one rig; they often fall short in most departments as a subsequent of attempting to do everything. Stability is always on my mind with these kind of things, especially with something that folds so small; a 20kg payload is re-assuring however. Having said that, LOKI will no doubt appeal to a certain kind of filmmaker; one that packs very light and perhaps more budget conscious. The latter is perhaps surprising as the LOKI isn't exactly cheap on first review, however when considering the costs of each individual configuration the cost starts to make sense. Check out the Kickstarter campaign here.Read more
by Kevin Alexander | 2nd March 2015
Edelkrone has announced updates to two of their products that are worth checking out. First, there is the new Pocket Rig 2, a compact camera rig ideal for run and gun scenarios. Pocket Rig 2 It may be a little bulky to actually fit in your pocket, but for good reason. A support arm folds out from underneath the baseplate. It's fully adjustable and allows you to comfortably shoot from the shoulder or even the hip. They've also included industry standard 15mm rods that unfold from the other side. You can easily attach a follow-focus and matte box. The rods even extend about 7 inches (18cm), providing ample space for all of your accessories. A standard 1/4 inch tripod connection hole allows the entire rig to be attached to a tripod. This would come in very handy in hectic environments when one must quickly alternate between handheld shooting and working off the tripod. I can definitely see how run and gun DSLR shooters will appreciate this tool, especially those who frequently travel. Larger rigs often take up a lot of space in gear bags, but this compact rig easily fits into a small camera bag. They also take a few minutes to set up. In a run and gun scenario those few minutes equal missed shots. When I'm shooting I don't always need a large rig, but I can't remember how many times I wished I had something smaller that gets the job done. The Pocket Rig 2 is an ideal fit for someone who doesn't need the bulk of a larger rig, and just wants something to help get that stable shot. And for $289 it's quite the deal. Pocket Skater 2 The second product Edelkrone has released is the Pocket Skater 2. Weighing in at only 1 pound, this little device is basically a compact dolly for a DSLR. But it doesn't just move in a straight line. It has 3 wheels that fold out and allow for curved slides. This little device really grabbed my eye. I like small pieces of gear that provide extra production value. When you look at a shot that used the Pocket Skater 2, you can't tell that it was produced with such a small device. You think it's shot with a dolly. The Pocket Skater 2 is priced at $249. Not bad for a dolly that fits in your camera bag. The Pocket Rig 2 is available for $289 HERE Pocket Skater 2 is available for $249 HERE POCKET RIG 2 TECH SPECS Folded Size: 5.7 x 2.3 x 2.3 inches (147 x 60 x 60mm) Weight: 1.5 pounds (.725Kg) Compatibility: All DSLR's & cameras with 1/4" – 20 thread (Optimized for DSLR's) Mount: 1/4" – 20 on top and 1/4" – 20 on bottom Support arm length: 11.4 inches (290mm) Locking mechanism: Constant friction, knob-less joints for hinge tensio n adjustment Materials: CNC machined aluminum, heavy duty construction and stainless steel screws POCKETSKATER2 Tech Specs Folded Size: 2.1 x 5 x 1.8 inches (55 x 127 x 47mm) Weight: 1 pound (.5 kg) Carrying capacity: Up to 5.5 pounds (2.5 kg) Mount: 1/4" – 20 on top Wheel radius: 1.5 inches (39 mm) Flex-Tilt Head Rotation Angle: 360 degrees Min. camera height (from ground to camera base): 3 inches (77 mm) Max. camera height (from ground to camera base): 5.5 inches (140 mm) Materials: CNC machined aluminum construction with delrin components; Stainless steel screws & rubber wheel ringsRead more
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4 Bedroom Detached House For Sale
Church Lane, Mablethorpe, LN12
Detached family home presented in exceptional condition throughout, gas central heating, extensive parking, double glazed windows, doors, soffits and fascias, fitted wardrobes in all bedrooms, lounge with a feature fireplace and gas fire, kitchen breakfast room with a...
3 Bedroom Semi Detached Bungalow For Sale
St. Andrews Road, Mablethorpe, LN12
Immaculate, extended, well presented dormer style bungalow with entrance hall, lounge with bay window, modern kitchen-diner, downstairs wc, downstairs double bedroom, sitting room, two double bedrooms upstairs and family bathroom. The property benefits from gas central
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Seaholme Road, Mablethorpe, LN12
Two bedroom park home with gas central heating and double glazed windows and doors, the accommodation comprises of a porch, lounge/diner, kitchen with a breakfast bar, study, conservatory, two bedrooms, bathroom with both a shower and bath, driveway and a garage...
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Field Coordinator/officer for Iitm Project Udc Sites in Bihar and Lakhimpur ( 1 - 6 yrs)
Pmanifold Business Solutions
RS 2,50,000 To 5,00,000 Bihar-Muzaffarpur, Gaya, Muzaffarpur, Sasaram, Lakhimpur, Lucknow
Job Purpose and Context field Coordinator/officer On Contract Basis for a New Technology Deployment Project Supported By Iit Madras, Ministry of Power and State Discoms this Position is for Project Udc. Udc is a Dc Based Power Technology Which is t...
Key Skills : sales,field,direct marketing,customer manager,district sales manager,direct sales executive,...
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Civil Engineer (huge & Urgent Requirment) ( 0 - 2 yrs)
Somya info edge services Pvt Ltd
RS 1,25,000 To 2,50,000 Gaya, Hajipur-Vaishali, Patna, North Delhi, Kannur, Muzhappilangad, Kolkata, Delhi
Position Description Designation: Manager - Technical Services Department: Technical Services Salary Range: Best In The Industry Years Of Experience: 0 To 3 Edu. Qualifications: Diploma - Civil Engineering. Other Details: * Ensurin...
Key Skills : civil engineer,project planner,brick work,planning executive,site engineer,site supervision,senior civil engineer,junior site civil engineer,...
AM/ Manager - Project Management / New Product Development ( 5 yrs)
Rucha Engineers
RS 8,00,000 To 13,00,000 Aurangabad - Bihar, Gaya
Prepare engineering analysis of projects,product approval from customer,Prepare technical specifications,7. Manage project issues, budgets, and schedules,Project Kick-off, Budget preparation, Pilot lot,machine tools, robots, automated mfg Equipment.
Key Skills : product development,,product planning,production engineer
Hiring Marketing Manager ( 2 yrs)
Axis Infotech
RS 1,50,000 To 2,50,000 Bhagalpur, Gaya, Patna, Purnia
- Identifying Existing and Potential Opportunities in the Company's Areas of Competence with Primary Focus On Corporate and Retail Segments - Conceptualizing Customized Audio Media Solutions for Clients - Preparing the Right Proposals, Meet, Nego...
Key Skills : business executive,area business manager,area sales manager,branch sales manager,area sales...