Regulation of Tryptophan Hydroxylase-2 Gene Expression by a Bipartite RE-1 Silencer of Transcription/Neuron restrictive Silencing Factor (REST/NRSF) Binding Motif

Abstract

Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in raphe serotonin biosynthesis, and polymorphisms of TPH2 are implicated in vulnerability to psychiatric disorders. Dynamic transcription regulation of TPH2 may underlie differences in vulnerability. We identified a transcription element in the TPH2 promoter that resembles the binding motif for RE-1 silencer of transcription (REST; also known as NRSF) transcription factor. REST limits tissue expression of non-neuronal genes through a canonical 21-bp motif called the NRSE (neuron-restrictive silencing element). The NRSE in TPH2 is a novel bipartite variant interrupted by a 6-base insertion. We confirmed that this bipartite NRSE permits transcriptional repression by REST identical to canonical NRSE in rat C6-glioma cells. Synthetic permutations of the motif revealed considerable flexibility in the juxtaposition of the two halves of bipartite NRSE. Computational analysis revealed many bipartite NRSE variants conserved between mouse and human genomes. A subgroup of these was found to bind REST by chromatin immunoprecipitation. Messenger RNAs for TPH2 and potassium channel H6, another gene with a bipartite NRSE, were up-regulated by dominant-negative REST in C6-glioma cells. These findings, which indicate that TPH2 expression is part of the developmental program regulated by REST and suggest that many previously unrecognized genes may be regulated by REST through the novel motif, have implications for the mechanism of REST action.Serotonin levels are regulated by the rate-limiting enzyme tryptophan hydroxylase, of which a neuronal isoform (NCBI Entrez Gene ID: TPH2) 3 was recently identified (1). TPH2 gene expression in the brain is restricted to raphe neurons, and TPH2 is likely the gene coding for the majority of tryptophan hydroxylase in these cells (1, 2). Serotonin modulates a variety of complex behaviors (3), and its signaling is implicated in the mechanism for a subset of antidepressants (4), but its precise role in mood regulation is unclear. A number of manipulations, including restriction of the essential dietary precursor tryptophan (5), stimulant drugs of abuse (6), or adverse developmental experiences (7), result in low brain serotonin levels and may underlie associated depression or anxiety phenotypes. Functional variants of TPH2 in mice are associated with decreased brain serotonin levels (8), and a TPH2 polymorphism in humans arguably (9) confers vulnerability to major depression (10). Associations with TPH2 polymorphisms are also reported for suicide (11), obsessivecompulsive disorder (12), and attention deficit disorder (13)(14)(15).In contrast to the growing number of genetic association studies, little is known about the regulation of TPH2 expression. Limited studies suggest that TPH2 mRNA levels are decreased following stress corticosteroids (16), unchanged (16) or increased by ovarian steroids (17), and increased by hypotensive stress (18) and one recent report suggests that POU3F2 may regulate TPH2 through a functional single-nucleoti...

Functional characterization of the human TPH2 5′ regulatory region: untranslated region and polymorphisms modulate gene expression in vitro

“…In the present study, the segment +8 to +53 of the TPH2 5′-UTR decreased luciferase activity and mRNA level strikingly, yet had a negligible effect on mRNA stability, suggesting that transcriptional mechanisms may be involved in the repression of gene expression by this region and that negative cis-element(s) should exist in this region. In accordance with these findings, a bipartite REST/NRSF binding motif that mediates transcriptional repression was recently identified in TPH2 5′-UTR between +9 to +35 (Patel et al 2007), just within the suppressive region (+8 to +53) revealed by our present study. In addition, our current preliminary data indicates that additional TF binding motifs in this region might also contribute to the transcriptional suppression of gene expression (data not shown).…”

“…1b, the proximal 5′ regulatory region (especially the inhibitory segment +8 to +53) shares a high homology between human, macaca and mouse TPH2. Putative TF binding sites for NEUROD1 (Beta-2/E47 dimer), Brn-2, and CEBP (CCAAT enhancer binding protein) were predicted in the core promoter, while the inhibitory region (+8 ∼ +53) contains putative sites for three TFs (NRSF, IA-1 and Myf-3), among which NRSF has been recently identified to confer transcriptional repression of TPH2 gene expression (Patel et al 2007), while IA-1 can also function as a transcriptional repressor (Breslin et al 2002). There are also several putative TF binding sites spanning or neighboring the 90A/G SNP, including two STRE (stressresponse element) sites.…”

“…Indeed, we have previously reported that functional polymorphisms in rhesus monkey TPH2 3′-UTR are strikingly associated with HPA axis function, whereas a common polymorphism 1952G/A (rs17110747) in the human TPH2 3′-UTR does not alter in vitro gene expression (Chen et al 2006). Recently, two papers regarding TPH2 gene expression regulation were published, of which one reported a functional significance of the −473T/A polymorphism (Scheuch et al 2007), while the other characterized a negative motif for REST/NRSF in the upstream of the 5′-UTR (Patel et al 2007). In the present study, we characterized cis-acting elements in the human TPH2 5′ regulatory region, as well as the functional significance of three previously described common SNPs in this region, including the two above-mentioned promoter SNPs (−703G/T and −473T/A) and another previously reported 90A/G SNP (rs11178998) in the 5′-UTR.…”

Inhibition of histone deacetylases enhances the function of serotoninergic neurons in organotypic raphe slice cultures

“…While the mechanisms that control TPH2 expression are not fully understood, several reports suggest that the neuron-restrictive silencer factor, which recruits corepressor complexes containing HDAC1/2 [18], negatively regulates TPH2 expression [19]. Consistent with these reports, the increase in TPH2 expression was accompanied by hyper-acetylation of histone H3 at the promoter region of the TPH2 gene in the raphe slice cultures.…”

The effect of long-term ovariectomy on midbrain stress systems in free ranging macaques

“…An unexplored mechanism involves repressor element silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF). REST is a repressor of transcription of TPH2 (Patel et al, 2007) and CRF (Seth and Majzoub, 2001). Hence, it is present in serotonin neurons.…”