The Drosophila gene snail (sna) which encodes a zinc finger protein is essential for dorsal-ventral pattern formation in the developing embryo. We have defined a repertoire of SNAIL (SNA) binding sites using recombinant SNA proteins to select specific binding sequences from a pool of random sequence nucleotides. The bound sequences which were selected by multiple rounds of gel retardation and amplification by the polymerase chain reaction (PCR) were subsequently cloned and sequenced. The consensus sequence, 5'G/A A/t G/A A CAGGTG C/t A C 3', with a highly conserved core of 6 bases, CAGGTG, shares no significant homology with known binding sequences of other Drosophila zinc finger proteins. However, the CAGGTG core is identical to the core motif of aHLH (helix-loop-helix) binding sites. The strongest SNA binding is obtained with sequences containing this core motif whereas reduced binding is seen for sequences with canonical CANNTG HLH motifs. Interestingly, SNA binding is detected in the promoter region of the snail gene. Transient expression in co-transfection experiments using a SNA binding element (SBE) linked to a heterologous promoter indicates that SNA has the ability to function as a transcription activator.
The rat pineal gland is a suitable model to investigate neurotransmitter-controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamine N-acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation of Aanat transcription are less clear. In this in vitro study we investigated the role of pCREB dephosphorylation for termination of Aanat gene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases in Aanat mRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE-stimulation for 8 h induced accumulation of PSP1-catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A-CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation of Aanat transcription in the rat pineal gland. Keywords: arylalkylamine N-acetyltransferase, ICER, norepinephrine, pCREB dephosphorylation, protein serine/threonine phosphatases, rat pineal gland. In many neuronal and neuroendocrine cells, neurotransmitter-driven signal transduction cascades converge on the activation of transcription factors that control cellular activity by initiating or repressing transcription of their target genes. The rat pineal gland represents an excellent model to study the regulatory mechanisms acting on the cAMP-controlled transcription factors CREB [cAMP responsive element (CRE)-binding protein] and ICER (inducible cAMP early repressor) and to link them with a defined output, the biosynthesis of the hormone melatonin (for review see Korf et al. 1998;Stehle et al. 2001).As in other systems, CREB is constitutively expressed in the pineal gland and its transcriptional impact depends on phosphorylation at serine 133, but not on de novo protein synthesis (Maronde et al. 1999a). Phosphorylation of CREB is induced by norepinephrine (NE; Roseboom and Klein 1995;Tamotsu et al. 1995;Korf et al. 1996), which is released exclusively during the night from sympathetic nerve endings and activates cAMP-dependent protein kinase A (PKA) type II via a b-adrenergic/cAMP mechanism (Korf et al. 1998;Maronde et al. 1999b). Phosphorylated (p) CREB enhances transcription of genes bearing a CRE in their promoter regions. Most importantly, NE elicits a more than 150-fold rise in the transcription of the gene encoding the arylalkylamine N-acetyltransferase (AANAT), the ratelimiting enzyme in melatonin biosynthesis (Borjigin et...
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