A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones.
The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.
Gerrard
Lesley
L
Roslin Institute, Roslin, Midlothian EH25 9PS, UK.
Howard
Mark
M
Paterson
Trevor
T
Thippeswamy
Thimmasettappa
T
Quinn
John P
JP
Haddley
Kate
K
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D050436:Regulatory Elements, Transcriptional; D015320:Tachykinins / Q000235:genetics* / Q000378:metabolism; D051720:Upstream Stimulatory Factors / Q000235:genetics / Q000378:metabolism
D011498:Protein Precursors; D015320:Tachykinins; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C044206:preprotachykinin; D020932:Nerve Growth Factor