Sp1 and Sp4 are bound in vivo to the GC-box region of TRPV1 P2-promoter
Search for TRPV1 genomic control elements capable of responding to inflammatory mediators revealed no classical response elements within the P2- promoter region [14
]. A search for alternative regulatory sites revealed tandem GC-box sites 5' to the P2 transcriptional start site (Figure , top). We have termed these two GC-box regions as: GC-box 'a' (GGGGAGGGGC) and GC-box 'b' (GGGAGGCCGGCC) (GenBank: DQ015702
). Since Sp1-like transcription factors are known to bind to GC-box sites and activate promoter regions in an NGF-dependent manner [18
], we first determined if any of the most well studied Sp1-like transcription factors could be expressed in rat DRG by performing a RT-PCR survey of mRNA for factors Sp1-4. mRNA encoding transcription factors Sp1, Sp3 and Sp4 but not Sp2 were identified in rat DRG (data not shown). To determine which of these Sp1-like transcription factors were expressed as protein in DRG and subsequently bound to the endogenous TRPV1 promoter region spanning GC-box "a" and "b", we utilized chromatin immunoprecipitation-ChIP analysis (Methods). ChIP provides for the in vivo
detection of candidate transcription factors bound to a known promoter region within its native chromatin structure [22
]. Although we were unable to develop primer sets that individually amplified GC-box "a" versus "b" due to the inherent GC-content and secondary structures (data not shown), it is understood that ChIP analysis provides a superior method (when compared to electrophoretic mobility shift assays - EMSA) to distinguish transcription factor binding that occurs only in the context of the native chromatin structure. This is especially critical given that Sp1-like factors (Sp1, Sp3, Sp4) are reported to have identical binding affinities to isolated GC-box binding targets when studied by EMSA in vitro
When sheared chromatin derived from intact DRGs harvested from rats 1.5 months of age or enriched cultures of neonatal DRG neurons were analyzed by ChIP (Methods), we successfully amplified DRG chromatin fragments using antisera directed against Sp1 or Sp4 (Figure ). Overall, strong evidence for Sp1 (3/4 ChIPs) and Sp4 (3/3 ChIPs) binding were observed (not all gels shown). In contrast, evidence for Sp3 binding was much less convincing (1/3 ChIPs) with a faint band representing the lowest levels of binding detectable amongst the three transcription factors tested (data not shown). When non-specific antiserum (IgG) was used for immunoprecipitation or when PCR amplification was performed without template DNA (primers alone - Pr), either a very faint band of smaller size was observed or no detectable fragment was visualized. Nevertheless, taken together ChIP analysis of rat DRG demonstrates transcription factors Sp1 and Sp4 binding to a region spanning GC boxes "a" and "b" within the P2-promoter. We then sought to understand what functional consequence Sp1-like factors have on TRPV1 promoter activity.
GC-box "a" is essential for activation of the TRPV1 P2-promoter
To establish a functional link between candidate GC-box sites and P2-promoter activation, we examined the effect of their individual deletion on P2-promoter activation in cultured DRG neurons and on NGF-dependent promoter activity in cultured PC12 cells. As shown in Figure , when the luciferase reporter construct 0.4 kb containing P2-promoter was transfected into cultured DRG neurons and luciferase activity was measured 48 hours later (Methods), a robust increase in transcriptional activity was observed when compared with the empty vector control. Following the selective deletion of GC-box "a", P2-associated promoter activity directed by the 0.4 kb reporter was completely lost. When GC-box "b" was deleted but GC-box "a" remained intact, there was a small decrease that did not reach significance. As shown in Figure , when identical experiments were conducted in an established model of NGF action - PC12 cells, we observed the previously reported NGF-dependent increase in P2-promoter activity following NGF treatment [14
]. However, we also observed the loss of NGF-dependent promoter activity with the deletion of GC-box "a" and a small decrease in NGF dependent activity with deletion of GC-box b that did not reach significance. When both GC-box "a+b" were deleted, the lowest observed level of promoter activity was obtained. These experiments suggest that GC-box "a", is essential for P2-promoter activation in DRG neurons as well as NGF-dependent transcription in PC12 cells. In contrast, GC-box "b", may have a modulatory role in DRG neurons given that its loss is associated with a trend to diminish promoter activity in DRG neurons.
Figure 2 GC-box "a" is essential for TRPV1 P2-promoter activation in DRG neurons and NGF-treated PC12 cells. Comparison of P2-promoter activity in DRG neurons + NGF (A) or +/- NGF-treated PC12 cells (B) directed by: empty pGL3 reporter plasmid (pGL-E); control (more ...)
The complete loss of promoter activity with deletion of GC-boxes "a+b" suggests that within the P2-promoter, no additional (cryptic) regulatory sites capable of promoter activation exist beyond GC-box "a & b". Given the evidence that transcription factor Sp1 is bound to the P2-promoter (Figure ), we then repeated this series of experiments under conditions of Sp1 over-expression. As shown in Figure , co-transfection of Sp1-cDNA with the P2-promoter construct 0.4 kb directed an increase in promoter activity. However, deletion of GC-box "a", or GC-box "a + b", again resulted in a complete loss of promoter activity whereas deletion of GC-box "b" did not show significant change in promoter activity. Similar results were observed in parallel experiments conducted in NGF treated PC12 cells (Figure ). An identical series of experiments was completed, now including conditions of Sp4 over-expression instead of Sp1 over-expression in cultured DRG neurons and NGF treated PC12 cells (Figure ). Although there was a trend for increased promoter activity under conditions of Sp4 over-expression in NGF treated PC12 cells, it did not reach significance. As previously observed under conditions of Sp1 over-expression, loss of promoter activity following deletion of GC-box "a" or GC-box "a + b" was not reversed by Sp4. Interestingly, deletion of GC-box "b" in this series was now associated with a statistically significant decrease in promoter activity in cultured DRG neurons and NGF treated PC12 cells.
Figure 3 GC-box "b" modulates TRPV1 P2-promoter activity. Comparison of P2-promoter activity in DRG neurons + NGF (A) or +/- NGF-treated PC12 cells (B) directed by: empty pGL3 reporter plasmid (pGL-E); control reporter plasmid (0.4 kb); 0.4 kb reporter with deletion (more ...)
Sp1 and Sp3 increase TRPV1 P2-promoter activity in cultured DRG neurons
Given evidence of Sp1, Sp4 and possibly small amounts of Sp3 - binding to the GC-box region, we then sought to determine what effect the over-expression of these Sp1-like factors would have on P2-promoter (0.4 kb) - directed promoter activity in cultured DRG neurons. As shown in Figure , we again observed the expected increase in promoter activity following transfection of the 0.4 kb construct [14
]. Co-transfection of expression plasmids encoding Sp1 or Sp3 expression plasmids with the 0.4 kb construct directed a further increase in promoter activity. On the other hand, co-transfection of the Sp4 expression plasmid did not show a significant increase. Interestingly, when Sp1 was paired with Sp3 or Sp4, no increase in promoter activity was observed, as was also observed when Sp3 was paired with Sp4. These results suggest that transcription factor Sp1 positively regulates TRPV1 P2-promoter activity and the presence of other members of the Sp1-like family (Sp3, Sp4) may serve to modulate or compete for control of transcription at the TRPV1 gene P2-promoter.
Figure 4 Transcription factors Sp1 and Sp3 increase P2-promoter activity in DRG neurons. P2-promoter reporter plasmid (0.4 kb) directs an ~8 fold increase in luciferase activity when compared with the empty reporter control (pGL-E). When construct 0.4 kb is co-transfected (more ...)
An inhibitor of Sp1-like transcription factors dose-dependently blocks NGF and Sp1- dependent TRPV1 promoter activity in PC12 cells
To further establish the role of Sp1-like transcription factors in the regulation of TRPV1 promoter activation, we then asked whether a known inhibitor of Sp1 function could disrupt P2-promoter activity in a model of NGF-dependent TRPV1 transcription. As previously observed [14
], NGF increased P2-promoter activity in PC12 cells (Figure , 0.4 kb black bars); however, mithramycin-a, an inhibitor of Sp1 function [26
] dose-dependently blocked the NGF-induced promoter activity. Importantly, mithramycin-a also dose-dependently blocked Sp1-dependent increases in P2-promoter activity (Figure ). Similar results were observed for Sp3 (data not shown). Attempts to perform identical experiments in neonatal DRG neurons were unsuccessful due to mithramycin-a associated toxicity and the requirement of NGF to sustain viability of neonatal DRG neurons, not seen with PC12 cells. Although the inhibitory effect of mithramycin-a does not preclude disruption of other Sp1-like member binding to GC-box binding sites, it does support the idea that in part NGF- dependent transcription at P2-promoter is mediated by Sp1 and/or other Sp1-like transcription factors.
Figure 5 An inhibitor of Sp1 (mithramycin-a) dose-dependently blocks NGF and Sp1- dependent P2-promoter activity in PC12 cells. P2-promoter reporter plasmid (0.4 kb) directs an ~2 fold increase in luciferase activity when treated with NGF × 48 hours [ (more ...)
siRNA directed knockdown of Sp1 decreases P2-promoter activity in DRG neurons and NGF-dependent activity in PC12 cells
Finally, we sought to demonstrate the dependence of endogenous Sp1 and Sp4 transcription factors on the activation of the P2-promoter through the use of a siRNA knockdown strategy previously shown to decrease Sp1 and Sp4 in primary cerebellar granule neurons [16
]. Search for off-site hits matched only the Sp1 and Sp4 sequence in a BLAST search of the NCBI nucleotide database (not shown). Although the low transfection efficiency using lipofectamine (≤ 5%) in DRG neurons precluded quantitative analysis of Sp1 or Sp4 content following siRNA treatment, the utility and fidelity of these probes have been previously reported [16
] and we have subsequently validated the efficacy of DNA constructs for over-expression or siRNA knockdown at the mRNA level following electroporation (see below). As shown in Figure , co-transfection of siRNA-Sp1 (Gift from G. Gill) into cultured DRG neurons significantly reduced P2-promoter activity directed by the 0.4 kb reporter plasmid. Co-transfection of siRNA-Sp4 showed a trend to decreased levels of promoter activity that did not reach significance. Similar findings were observed when promoter activity was studied in transfected PC12 cells. As shown in Figure , NGF again directed an expected increase in 0.4 kb reporter activity whereas co-transfection of Sp1-siRNA decreased the NGF-dependent promoter activity. Moreover, under conditions of Sp1 over-expression (Figure , 0.4 kb Sp1), the additional increase in promoter activity directed by Sp1 was significantly reversed by co-transfection of Sp1-siRNA. In addition, co-transfection of siRNA-Sp4 also produced a decrease in promoter activity in NGF-treated PC12 cells.
Figure 6 P2-promoter activity in DRG neurons is decreased by Sp1-siRNA and Sp1 and Sp4 siRNA also block P2 promoter activity in NGF treated PC12 cells. Co-transfection of Sp1-siRNA with the 0.4 kb P2-promoter construct resulted in a significant decrease in promoter (more ...)
Over-expression of Sp1 or Sp4 increase endogenous levels of TRPV1 mRNA in cultured DRG neurons
Building on our observations that Sp1 and Sp4 are bound to the TRPV1 P2-promoter region in vivo and regulate P2-promoter activity, we then attempted to manipulate Sp1 or Sp4 expression in cultured DRG neurons to determine their subsequent downstream effects on changes in endogenous TRPV1 mRNA expression. Given that our lipofectamine-based transfection of DRG neurons and PC12 cells provide relatively small ( < 5%) transfection efficiencies (Methods), we elected to conduct these experiments in cultured DRG neurons following electroporation (Methods) to provide greater transfection rates (30-40%) based on GFP staining in viable cells at 24-48 hours (not shown). As shown in Figure , we first quantified endogenous levels of rat Sp1 mRNA in cultured DRG neurons following transfection with the empty expression plasmid PN3. Because no amplification of the Sp1/Sp4 genes for the reference control sample occurs and 2--ΔΔCt analysis cannot be utilized, CT values are used instead of RQ values to compare mRNA expression levels (Methods). We then measured the resultant mRNA content of human Sp1 mRNA following either electroporation with the empty PN3 vector versus a human Sp1/PN3 expression plasmid (previously used in promoter activity assays) and compared its CT value with the CT value associated with baseline levels of the rat Sp1 or Sp4 mRNA.
Figure 7 Over-expression of transcription factor Sp1 and Sp4 mRNA increase endogenous TRPV1 mRNA in cultured DRG neurons. (A) Measurement of endogenous levels of rat Sp1 mRNA in cultured DRG neurons following transfection with empty/PN3 vector (left). Additional (more ...)
Human Sp1-like transcription factors and their cognate cDNAs differ slightly in nucleotide sequence, but encode indistinguishable functional properties across species. Therefore, we were able to independently measure and compare the additional contribution of human Sp1 mRNA. As shown in Figure , following transfection with the human Sp1 cDNA, an approximately equal amount of human Sp1 mRNA in addition to the endogenous rat Sp1 mRNA was detected in cultured DRG neurons. Therefore, following an approximate doubling of Sp1 mRNA, we observed a significant increase in endogenous TRPV1 mRNA (Figure ). In like manner, we repeated these experiments but measured the endogenous expression of rat Sp4 mRNA (Figure ) and subsequently human Sp4 mRNA content in cultured DRG neurons following electroporation of the Sp4 cDNA. Again, we observed an approximate doubling of Sp4 mRNA with a corresponding significant increase in TRPV1 mRNA (Figure ).
Double knockdown of transcription factors Sp1 and Sp4 directs a decrease in TRPV1 mRNA in cultured DRG neurons
Having observed the generally positive regulatory effects of Sp1 and Sp4 on TRPV1 mRNA expression in cultured DRG neurons (Figure ), we returned to a siRNA knockdown strategy to help confirm the relationship between Sp1, Sp4 and TRPV1 RNA transcriptional control. As shown in Figure , following electroporation of cultured DRG neurons with Sp1-siRNA, a significant decrease in Sp1 mRNA was detected when compared with control experiments conducted with a scrambled Sp1-like siRNA control vector. However, no significant changes were observed in concurrently measured Sp4 mRNA or TRPV1 mRNA content. When parallel experiments with siSp4 - mediated knockdown were conducted, a decrease in Sp4 mRNA was observed. Importantly, when Sp4 mRNA knockdown was achieved, there was evidence of a concurrent decrease in Sp1 and TRPV1 mRNA (Figure ). Finally, given the apparent "cross-talk" between Sp1 and Sp4 gene expression, we electroporated an equal ratio (1:1) of siSp1 plus siSp4 and observed a significant decrease in TRPV1 mRNA in cultured DRG neurons (Figure ).
Figure 8 Knock-down of transcription factors Sp1/Sp4 decrease endogenous TRPV1 mRNA in cultured DRG neurons. (A) Evidence of Sp1mRNA knockdown following transfection of siSp1 in cultured DRG neurons (***) p < 0.0001 (left). Apparent changes in Sp4 (middle) (more ...)