Quantitative effects on gene silencing by allelic variation at a tetranucleotide microsatellite.

Microsatellites are common repeated sequences, which are useful as genetic markers and lack any clearly established function. In a previous study we suggested that an intronic polymorphic TCAT repeat in the tyrosine hydroxylase (TH) gene, the microsatellite HUMTH01, may regulate transcription. The TH gene encodes the rate-limiting enzyme in the synthesis of catecholamines, and the microsatellite HUMTH01 has been used in genetic studies of neuropsychiatric and cardiovascular diseases, in which disturbances of catecholaminergic neurotransmission have been implicated. HUMTH01 alleles associated with these diseases act as transcriptional enhancers when linked to a minimal promoter and are recognized by specific nuclear factors. Here we show that allelic variations of HUMTH01 commonly found in humans have a quantitative silencing effect on TH gene expression. Two specific proteins, ZNF191, a zinc finger protein, and HBP1, an HMG box transcription factor, which bind the TCAT motif, were then cloned. Finally, allelic variations of HUMTH01 correlate with quantitative and qualitative changes in the binding by ZNF191. Thus, this repeated sequence may contribute to the control of expression of quantitative genetic traits. As the HUMTH01 core motif is ubiquitous in the genome, this phenomenon may be relevant to the quantitative expression of many genes in addition to TH.

CREM and ICER are differentially implicated in trans-synaptic induction of tyrosine hydroxylase gene expression in adrenal medulla and sympathetic ganglia of rat.

Reserpine treatment leads to a trans-synaptic increase of the tyrosine hydroxylase (TH) gene transcription rate, mRNA and protein levels in catecholaminergic tissues including the adrenal medulla (AM) and the superior cervical ganglia (SCG). The TPA-responsive element plays an important role in the trans-synaptically-induced transcription of the TH gene in the AM, whereas it does not appear to be involved in the SCG (Trocmé et al. [1997] J. Neurosci. Res. 48:489-498). In this study, we show that another regulatory sequence of the TH proximal promoter, the cAMP-responsive element (CRE), binds different factors in the AM and in the SCG. To elucidate the dynamics of promoter regulation a complete time course analysis was conducted. Reserpine treatment enhances, between 1 hr and 8 hr after the injection, the expression and the binding of the repressor ICER in the AM, whereas in the SCG it enhances the binding of CREM factors. These results suggest that the mechanisms mediating trans-synaptic induction of the TH gene are different in the AM and SCG. The interplay between positive and negative transcription factors and their kinetics of action are responsive of the long-term regulation of the TH gene.

Multiple complexes involved in tyrosine hydroxylase mRNA stability in rat adrenal medulla, after reserpine stimulation.

A 28-nucleotide sequence within the 3'-untranslated region (3'UTR) of tyrosine hydroxylase (TH) mRNA has been suggested to influence the turnover rate of the TH messenger in vitro (W. R. Paulding and M. F. Czyzyk-Krzeska, 1999, J. Biol. Chem. 274, 2532-2538). In this study, we show that treatment with reserpine, a catecholamine-depleting drug which increases the stability of TH mRNA, allows the binding of a cytosolic protein to this 28-mer site in the TH 3'UTR in the rat adrenal medulla. An ex vivo kinetic analysis shows that the resulting 54-kDa ribonucleoprotein is early induced by reserpine. However, the formation of this complex is not coupled with the upregulation of TH mRNA, indicating that this 54-kDa complex could not be the unique factor accountable for the long-term stabilization of the TH messenger. Following this result we found that several other cis-acting elements, located in single-stranded stem loops within the secondary structure of TH 3'UTR, formed multiple complexes (43, 54, and 105 kDa) with cytosolic, polysome-associated, and also nuclear proteins. Our findings demonstrate that the messenger stability does not depend solely on the formation of a unique RNA-protein complex, but involves mechanisms with higher complexity implicating the interactions between posttranscriptional, nuclear RNA export, and translational processes.

Tyrosine hydroxylase in the european eel (Anguilla anguilla): cDNA cloning, brain distribution, and phylogenetic analysis.

We report the isolation of a full-length eel tyrosine hydroxylase (TH) cDNA that is characterized by a long 3' untranslated region and by a diversity restricted to the 3' end owing to the differential use of three polyadenylation signals. The longest eel TH mRNA was distinctive in the presence of four pentameric elements (AUUUA) in the AU-rich 3' noncoding region. Such a diversity could provide the basis of posttranscriptional or translational regulation of eel TH gene expression. Comparison of the eel TH sequence with those of other aromatic amino acid hydroxylases (TH, tryptophan hydroxylase, and phenylalanine hydroxylase) and phylogenetic analysis confirmed that the N-terminal regulatory domain is highly divergent, contrasting with the conservation of the catalytic core of the enzyme. Molecular phylogenies including the available sequences of the three hydroxylase genes suggested that the duplication of their common ancestor occurred before the emergence of arthropods. The regional expression of the eel TH mRNA was studied by semiquantitative PCR, northern blots, and in situ hybridization and compared with the immunocytochemical localization of TH protein. The data showed that TH mRNA is mostly expressed in the olfactory and hypothalamic areas, whereas sparse TH-expressing cell bodies are present in the telencephalic region and brainstem. No labeling was detected in the mesencephalic area, in striking contrast with that found in amphibians and amniotes.

Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation.

Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.

AP-1 mediates trans-synaptic induction of tyrosine hydroxylase gene expression in adrenal medulla but not in superior cervical ganglia.

Reserpine treatment leads to a rapid trans-synaptic increase of the tyrosine hydroxylase (TH) gene transcription rate and mRNA levels in catecholaminergic tissues including the adrenal medulla (AM) and the superior cervical ganglia (SCG). In the AM, the formation of a specific protein complex with the TPA-responsive element located in the proximal region of the TH gene was enhanced between 30 min and 8 hr following the injection. This complex appears to contain a member of the Fos family and an antigenically related Jun protein. Moreover, the prolonged and enhanced expression of the c-Fos protein in the AM and its phosphorylation are likely to contribute to the increased TH transcription following reserpine treatment. Most strikingly, in the SCG, the trans-synaptic induction of TH transcription is transduced by totally different mechanisms, since no AP-1 complex and only minute amounts of c-Fos immunoreactivity were detected. Our study provides the first demonstration that, following the same stimulus, the induced expression of a single gene is mediated by different cis- and trans-acting factors in two distinct tissues sharing the same embryonic origin.

Multiple tyrosine hydroxylase transcripts and immunoreactive forms in the rat: differential expression in the anterior pituitary and adrenal gland.

Several regulatory neurofactors, classically associated with the hypothalamus, may be synthesized in the anterior pituitary (AP). Dopamine (DA) is the main prolactin-inhibiting factor. Its de novo synthesis in the normal AP has not been proved, although the TH transcript has been previously demonstrated by RT/PCR in the AP. We investigated tyrosine hydroxylase (TH) gene expression at both the protein and mRNA levels in the AP of normal random cycling female rats and in a catecholaminergic tissue, the adrenal gland (AG). The Western blot analysis of AP homogenates revealed two immunoreactive forms of TH in the AP, both differing from the TH present in the AG. RT/PCR products from AP and AG mRNA were subcloned and sequenced. In addition to the full-length form, we identified two TH transcripts generated by alternative splicing either involving the use of a new alternate splice-donor site within exon 2 or skipping exon 11. The form lacking exon 11 was not isolated from the AG. In the AP, all three forms were present. Although the AP contained the full-length TH mRNA, the expected size protein was not detected. Thus, there is alternative splicing of the TH primary transcript, and putative additional post-translational regulation may yield TH proteins with no enzymatic activity, at least in non-catecholaminergic tissues.

Expression of human dopamine beta-hydroxylase in mammalian cells infected by recombinant vaccinia virus. Mechanisms for membrane attachment.

Dopamine beta-hydroxylase (DBH) is found in neurosecretory vesicles in both membrane-bound and soluble forms. We expressed various human DBH cDNAs in two mammalian cell lines, using the vaccinia virus expression system. The expression of a full-length DBH cDNA (DBH-f) reproduced the native DBH electrophoretic pattern and led to the synthesis of an active enzyme composed of two subunits of 77 and 73 kDa. In contrast, a truncated cDNA lacking the first ATG (DBH-t) generated a single band of 73 kDa. Analysis of mutated recombinant clones demonstrates that the two polypeptides do not result from the use of an alternative translation initiator codon. These results, combined with deglycosylation experiments, allow us to attribute the double band pattern to an optional cleavage of the signal peptide. When the NH2-terminal extremity is shortened, cleavage becomes obligatory, underlining the role of the first 14 amino acids in the regulation of the cleavage of the signal peptide. Subcellular analysis of recombinant DBH-t and DBH-f proteins indicates that DBH is anchored to the membrane by two distinct mechanisms; one of them is due to the non-removal of the signal peptide, whereas the second one is independent of the presence of the signal sequence. Moreover, quantification of the fractionation experiments suggests that the two modes of membrane attachment are additive.

TH mRNA over-expression in rats with chronic excitotoxic striatal lesions.

Twenty weeks after ibotenic acid lesions of the striatum, the amount of tyrosine hydroxylase (TH) in this structure was markedly increased. This was accompanied by a 3-fold increase in TH mRNA levels in the ipsilateral subtantia nigra (SN). Striatal levels of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) were markedly reduced. In the nucleus accumbens, spared by the lesion, DA neurotransmission was also altered, as evidenced by a reduction of DA and DOPAC, but no increase in TH could be detected. TH mRNA levels were moderately enhanced in the ventral tegmental area (VTA). Thus, lesioning in the striatum induces TH gene activation in both SN and VTA neurones, not strictly related to DA function at the terminal level.

Regulation of tyrosine hydroxylase gene expression in mesencephalic dopamine neurons: effect of imipramine treatment.

The effects of a chronic imipramine treatment on the mesoamygdaloid pathway of rats were examined. Using semiquantitative immunocytochemical techniques, it was observed that the level of TH mRNA was decreased in the ventral tegmental area (VTA). In contrast, the TH protein was increased in both the VTA and amygdala. The TH activity was decreased in the amygdala when assessed under normal conditions but increased after a preincubation to phosphorylate the enzyme, suggesting a lowering of the protein-specific activity in the terminals. These results show that TH protein turnover in the mesoamygdaloid neurons can be reduced by chronic imipramine treatments, thereby producing an accumulation of inactive TH protein in the neurons while also decreasing TH gene activity in the cell bodies.

Induction of calbindin-D 28K gene and protein expression by physiological stimuli but not in calcium-mediated degeneration in rat PC12 pheochromocytoma cells.

To understand the role of calbindin-D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin-D 28K is expressed in PC12 cells. The amounts of calbindin-D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D3. Calbindin-D 28K expression was also significantly up-regulated by stimuli (depolarization, low concentrations of Ca2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin-D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin-D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.

Analysis of the human dopamine beta-hydroxylase promoter: transcriptional induction by cyclic AMP.

We have analyzed some functional aspects of the promoter of the human dopamine beta-hydroxylase (DBH) gene. A fragment of 1,247 bp directly 5' to the transcriptional start was progressively shortened, placed in front of a reporter gene, and tested in a human neuroblastoma cell line expressing DBH (SK-N-SH-TFM) and in a monkey kidney cell line (CV-1). A remarkably short region (267 bp), directly upstream from the transcription start, was sufficient to confer activity and tissue-specific expression. Furthermore, the expression of the DBH gene was shown to be inducible by cyclic AMP in SK-N-SH-TFM cells. This effect was demonstrated to occur at the transcriptional level, as shown by run-on assays, and was due to the presence of a near-consensus cyclic AMP-responsive element located in the untranscribed 5' regulatory region of the gene.

Evidence of tyrosine hydroxylase mRNA in the anterior and neurointermediate lobes of female rat pituitary.

The anterior pituitary is thought to be unable to synthesize dopamine (DA) except under experimental conditions where a tyrosine hydroxylase (TH) activity, the rate-limiting step of its synthesis, has been demonstrated. In this work, we tested whether the enzyme described as active under particular conditions comes from de novo TH gene transcription or from a pre-existing TH mRNA poorly translated or untranslated under physiological conditions. Therefore, we searched for the presence of TH mRNA in normal female rat pituitary using the polymerase chain reaction following reverse transcription (RT/PCR) and in situ hybridization (ISH). The neurointermediate lobe (NIL) of the hypophysis was used as negative tissue, since it is thought to be unable to synthesize TH. As expected, no ISH labelling could be seen in the neural lobe (NL). However, scarce labelled cells were found in the intermediate lobe (IL) confirming the positive results observed in the NIL by RT/PCR. The anterior lobe (AL) also presented TH mRNA by PCR and ISH. The TH gene expression in sparse cells of the AL is discussed in regard to the ability of the AL to synthesize DA under particular conditions from a pre-existing mRNA.

AP-1 complex and c-fos transcription are involved in TPA provoked and trans-synaptic inductions of the tyrosine hydroxylase gene: insights into long-term regulatory mechanisms.

We have previously shown that the phorbol ester, TPA, which activates protein kinase C, causes, in PC12 cells, a transcriptional activation of tyrosine hydroxylase (TH), the key enzyme in catecholamine synthesis. The study has now been extended to examine the processes that underlie this transcriptional stimulation and, in addition, to seek whether similar mechanisms are involved in long-term trans-synaptic induction of the TH gene in adrenal medullae of rats that have been given a single injection of reserpine. In both systems, it was found that the induction of c-fos gene transcription was associated with that of the TH gene but with different kinetics. The promoter of the TH gene contains (at position -207/-200) a sequence (TGATTCA) which differs from the consensus TRE or AP-1 site (TGACTCA) by one nucleotide. Experiments were carried out to investigate whether the AP-1 protein complex which is known to contain Fos and Jun binds to the putative TRE region of the TH promoter. In the gel shift assays, the nuclear protein extracts derived from TPA-treated PC12 cells and from AM of reserpine injected rats displayed a higher magnitude of binding to a 25-mer TRE-TH oligonucleotide as compared to controls. The results showed that the behaviour of TRE-TH was atypical in that two retarded complexes (A and B) were observed, which were displaced by specific competitors. Trans-activation experiments with plasmids TRE-TH/TK/CAT and -754/-19 TH/pUC18-CAT in PC12 cells showed an increase in CAT activity in response to TPA that correlates with the previously observed increase in TH transcriptional activity by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression of tyrosine hydroxylase in the developing fetal brain.

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.

Short- and long-term alterations of gene expression in limbic structures by repeated electroconvulsive-induced seizures.

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.

Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion.

Increased synaptic stimulation of sympathetic neurons in vivo causes a delayed increase in the activity and the amount of tyrosine hydroxylase (TH). To determine whether these changes result from an increase in the messenger RNA for TH, the rat preganglionic cervical sympathetic trunk was electrically stimulated unilaterally for 90 min, and 48 h later RNA was extracted from stimulated and contralateral control superior cervical ganglia. Northern blots probed with a cDNA for TH demonstrated that nerve stimulation produced about a 2.5-fold increase in the amount of TH mRNA in the ganglion. These results indicate that synaptic stimulation leads to an increase in TH mRNA, either by increasing the rate of transcription of the TH gene or by increasing the stability of its mRNA.

Modulation of GFAP mRNA levels following toxic lesions in the basal ganglia of the rat.

GFAP mRNA levels were quantified by Northern blot analysis using a human GFAP (glial fibrillary acidic protein) cDNA probe in association with immunocytochemistry. Ten days after a unilateral lesion of substantia nigra with 6-hydroxydopamine (6-OHDA), GFAP mRNA level is increased 1.4-fold in the ipsilateral striatum; thereafter it declined continuously to reach the control level 4 months later. This effect contrasted with the lower and more sustained increase of preproenkephalin (PPE) mRNA, a marker of neuronal target of nigrostriatal pathway. Following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, we observed a sharp elevation of the GFAP transcripts (4-fold) as soon as 2 days after the lesion both in the striatum and in the substantia nigra. Whereas in the striatum GFAP mRNA level already declined at 5 days postlesion, it remained stable in the substantia nigra. In comparison GFAP immunoreactivity was slightly delayed. No obvious modification was observed in the contralateral side to the lesion whatever the denervation condition studied. Implication of these results on the understanding and the therapeutic approach of glial scarring is discussed.

Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization.

A rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as a control probe. Using the tyrosine hydroxylase probe, we ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of 35S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

Synthesis of catalytically active choline acetyltransferase in Xenopus oocytes injected with messenger RNA from rat central nervous system.

Choline acetyltransferase (ChAT) mRNA and ChAT enzymatic activity have been compared in different regions of the rat central nervous system. mRNA was assayed by exploiting the Xenopus oocyte system which was first tested by measuring the electrophysiological response to glycine after injection of mRNA derived from the ventral part of the spinal cord (VSC). This tissue was found to contain the highest ChAT mRNA level. The striatum, which yielded the maximal enzymatic activity, contained 10 times less ChAT mRNA than the VSC. These results are discussed in terms of the neuroanatomical differences between the two structures.