TRANSCRIPTIONAL ACTIVATION OF HUMAN CHOLINE-ACETYLTRANSFERASE BY AP2-INDUCED AND NGF-INDUCED FACTORS

ChAT (choline acetyltransferase) is the enzyme responsible for acetylc holine synthesis and is specifically expressed in cholinergic neurons. To further characterize the transcriptional regulation of the hCHAT ( human ChAT) gene by NGF, we examined the effects upon ChAT promoter ac tivity of a family of transcription factors which are activated by NGF and several extracellular stimuli and encoded by immediate-early gene s. These include NGFI-A (Egr1, zif268), NGFI-C (Egr2), Krox-20 and NGF I-B (Nurr77). Two fragments of the hChAT gene were used for functional analysis carrying 944 bp (pi) and 4000 bp (pi + P2) of the 5' flankin g region in front of the chloramphenicol acetyltransferase (CAT) repor ter gene. They were transiently co-transfected with NGFI-A, NGFI-C, Kr ox-20 and NGFI-B expression vectors in NG108-15, SN6 and COS-1 cells. CAT activity after transfection of the p4000 ChAT-CAT reporter into bo th neuronal cell lines (NG108-15 and SN6 cells) was increased up to 5- fold in the presence of co-transfected NGFI-A and up to 5- and 12-fold after co-transfection of NGFI-C expression vector in NG108-15 and SN6 cells, respectively. Tn NG108-15 cells, dbcAMP excerted a strong enha ncing activity on the transactivation properties of NGFI-C while this was not observed when cells were transfected with NGFI-A. These trans- activation effects were specific for neuronal cells. When NG108-15 cel ls were treated with dbcAMP in the presence of H89, a specific PKA inh ibitor, the increase of transcriptional activity of NGFI-C was abolish ed, indicating that a signalling transduction mechanism through PKA pl ays a role in NGFI-C-induced trans-activation, Electrophoretic mobilit y-shift assays showed that the sequence GCCCGGGGAG (NGFRE) located 120 5 bp upstream of the first coding ATC (El) can bind NGFI-A but not NGF I-C. Several possibilities explaining the observed results are discuss ed. Finally, transfections of ChAT-CAT reporters including the pi + P2 region or a minimal ChAT enhancer present in the P2 region in front o f a heterologous promoter indicated the presence of a regulatory eleme nt which conferred AP2-dependent trans-activation with homologous as w ell as with heterologous promoter constructs. (C) 1997 Elsevier Scienc e B.V.