Novel Dual Repressor Elements for Neuronal Cell-specific Transcription of the Rat 5-HT1A Receptor Gene

Abstract

The serotonergic neurons of the raphe nuclei are the primary site of serotonin synthesis in the brain. They send projections to a wide variety of brain regions, including the hippocampus, cortex, limbic system, and hypothalamus (1). Activation of postsynaptic receptors in the above regions is associated with serotonergic regulation of memory, motivation, emotion, neuroendocrine stress response, etc. (2-4). The activity of serotonergic neurons of the raphe nuclei is regulated in part by presynaptic autoreceptors. The 5-HT1A autoreceptor is located at the cell body and dendrites of raphe serotonergic neurons (5, 6) and mediates negative feedback inhibition of the firing rate through recurrent activation of potassium channels via pertussis toxin-sensitive G proteins (7) to decrease serotonin release. Thus the 5-HT1A receptor plays a major role in controlling serotonergic outflow to the wide variety of brain regions that are innervated by the raphe nuclei.Abnormal regulation of 5-HT1A receptor expression is implicated in depression and anxiety disorders. 5-HT1A receptor knockout mice display increased anxiety-related behaviors (8 -10), suggesting that a loss of the 5-HT1A autoreceptors is correlated with symptoms of anxiety (3). On the other hand, 5-HT1A receptor levels are increased in the midbrain of suicide victims with major depression compared with nondepressed suicides (11). Down-regulation of the 5-HT1A autoreceptor by antidepressants (12, 13) disinhibits action potential firing of the raphe neuron, thereby enhancing serotonergic neurotransmission (14 -17). The prolonged (2-3 week) time course required for antidepressant action suggests an alteration in transcriptional activity of the 5-HT1A receptor.To investigate the mechanisms that regulate cell-specific and basal regulation of the 5-HT1A receptor, we have identified the transcriptional start site and examined the regulation of the transcriptional activity of a 2.719-kb 1 fragment of the rat 5-HT1A receptor gene in several cell lines, including 5-HT1A receptor-positive RN46A raphe and SN48 septal cells (18 -20) and receptor-negative L6 myoblast and C6 glioblastoma cells (21). We identified a region of the rat 5-HT1A receptor gene located upstream of an ubiquitously active promoter region that reduces transcriptional activity. In the present study, we have identified a 14-bp element in the 5Ј-flanking region of the 5-HT1A gene that mediates transcriptional repression in raphe cells, but is dispensable in receptor-negative cells where an adjacent 12-bp element maintains repression of the gene. In contrast to the single repressor-DNA complex present in 5-HT1A receptor-expressing cells that may modulate basal levels of receptor expression, the presence of two protein-DNA complexes in receptor-negative cell lines provides a dual mechanism to repress 5-HT1A receptor expression.

Increased Serotonin-1A (5-HT1A) Autoreceptor Expression and Reduced Raphe Serotonin Levels in Deformed Epidermal Autoregulatory Factor-1 (Deaf-1) Gene Knock-out Mice

“…The 5-HT1A receptor gene is regulated by a proximal promoter and an upstream repressor region that determines basal expression in neuronal cells (14,15). In the human 5-HT1A repressor region, we previously identified a functional C(Ϫ1019)G polymorphism associated with major depression and suicide, as well as anxiety-related phenotypes, which has been replicated in independent studies (16,17).…”

“…Briefly, sense and antisense oligonucleotides of the human (26C) and mouse (mouse#1, 5Ј-GGAGAGTCTCTGAGGGTTTTCCTCGTGC-CTGATA) Deaf-1 elements synthesized with GG 5Ј-overhangs were hybridized and labeled with [ 32 P]dCTP using Klenow fragment DNA polymerase (15). Purified bacterially expressed human Deaf-1 protein (5 or 7.5 g/reaction) was preincubated with or without competitor DNA in a 20-l reaction containing labeled probe in DNA-binding buffer (20 mM HEPES, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5% glycerol, and 2 mM DTT, pH 7.9), 1 g of poly(dI-dC), at 22°C for 20 min and resolved on a nondenaturing 5% acrylamide/Tris-glycine gel at 4°C.…”

Transcriptional Mechanisms for Induction of 5-HT1AReceptor mRNA and Protein in Activated B and T Lymphocytes

“…Treatment of transfected Chinese hamster ovary cells with 5-HT 1A agonists has been shown previously to increase 5-HT 1A receptor density via activation of the NF-B pathway, by stimulating the degradation of the inhibitory subunit, I-B (32). Two consensus NF-B binding sites (at Ϫ64 and Ϫ365 bp upstream from the initiation ATG) are located in a region with strong enhancer activity that is highly conserved in rat and mouse (33)(34)(35). In addition, recent studies have shown that the p50/p65 subunits of NF-B are positive regulators of the rat 5-HT 1A receptor promoter activity (36).…”

Transcriptional regulation of the 5-HT1Areceptor: implications for mental illness

“…These include the RE-1 site for neural restrictive factor (REST/NRSF) [76] and a powerful dual repressor element that is regulated by a pair of conserved repressors, Freud-1/ CC2D1A and Freud-2/CC2D1B [77][78][79]. Unlike REST, which silences neuronal genes mainly in nonneuronal cells, Freud-1 and Freud-2 also repress 5-HT 1A expression in neuronal cells [76].…”