Characterization of rat and human tyrosine hydroxylase genes: Functional expression of both promoters in neuronal and non-neuronal cell types

doi:10.1016/S0006-291X(88)81022-2

Biochemical and Biophysical Research Communications

Volume 157, Issue 3, 30 December 1988, Pages 1341-1347

https://doi.org/10.1016/S0006-291X(88)81022-2 Get rights and content

We describe the structure of the promoter and intron 1 of the human and rat tyrosine hyroxylase genes. The 5′ flanking region of the two genes are 74% identical (+1 to −380) and contain a completely conserved cAMP response element. Although both genes are single copy, multiple splicing events of the human transcript lead to multiple mRNAs. Based on several lines of evidence alternative forms of mRNA of rat TH analogous to those in the human are not present. Both rat and human promoters direct the transcription of reporter genes when introduced into rat pheochromocytoma and fibroblast cells.

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Cited by (51)

Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo
2016, Molecular Therapy Methods and Clinical Development
Citation Excerpt :
TagRFP-T DNA was amplified and cloned into an AAV backbone with the chicken β-actin promoter, and a woodchuck postregulatory element (WPRE) at the 3′ end (pBL). Three partial sequences of the hTHp were based on GenBank Acc# AY211521: 522bp (spanning from −491 to +31),4 a combination of 5′-flanking region (spanning from −509 to +1) and a 976 bp segment of 3′-flanking region DNA extending in the 3′ direction from the PolyA signal,6 and 3.3 kb (spanning from −3204 to +3)7 were produced by polymerase chain reaction amplification from human genomic DNA (Promega, Madison, WI) and subcloned in pGEM-T vector system (Promega). The following primers pairs were used: for the 522bp sequence, 5′-AGACACACGGCCTGGAATCT-3′ (forward), 5′-CCTGTGGCGTGGTGGCGTCGGGGGTGGGCATGGCTCAGTGTGGA-3′ (reverse); and for the 3.3 kb sequence,5′-GACCAGTGTCTTGGGAGTTG-3′ (forward), 5′-CATGGCTCAGTGTGGAGGTCCGG-3′ (reverse).
The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.
Epigenetic, transcriptional and posttranscriptional regulation of the tyrosine hydroxylase gene
2011, International Journal of Developmental Neuroscience
Citation Excerpt :
Most of these elements are not conserved. Five subregions located in the region from −9 kb to −2 kb (Kessler et al., 2003) and elements present in the first 200 nucleotides upstream of the nucleotide +1 are an exception, and the latter preserve both the regulatory elements and their locations (Harrington et al., 1987; Coker et al., 1988; Kobayashi et al., 1988; Cambi et al., 1989; D’Mello et al., 1989; Iwata et al., 1992, Fig. 1). The existence of only a small sequence homology in the promoter region of the mouse, rat, and human TH genes indicates species specificity of some regulatory elements (Romano et al., 2005).
The activity of tyrosine hydroxylase (TH, EC 1.14.16.2) gene and protein determines the catecholamine level, which, in turn, is crucial for the organism homeostasis. The TH gene expression is regulated by near all possible regulatory mechanisms on epigenetic, transcriptional and posttranscriptional levels. Ongoing molecular characteristic of the TH gene reveals some of the cis and trans elements necessary for its proper expression but most of them especially these responsible for tissue specific expression remain still obscure. This review will focus on some aspects of TH regulation including spatial chromatin organization of the TH locus and TH gene, regulatory elements mediating basal, induced and cell-specific activity, transcriptional elongation, alternative TH RNA processing, and the regulation of TH RNA stability in the cell.
Gene therapy for dopamine replacement
2010, Progress in Brain Research
Citation Excerpt :
Truncation of parts of the N-terminal domain appear to mimic the full phosphorylation in regard to increasing TH enzyme activity and increasing the Ki of dopamine (Kuczenski, 1973; Musacchio et al., 1971; Vigny and Henry, 1981). The idea to restore striatal dopamine production by gene transfer of the dopamine synthetic enzymes goes back to the late 1980s, constituting a very productive new line of research where the TH gene was overexpressed in cultured cells (Coker et al., 1988; Horellou et al., 1989). In majority of the studies, the cell type of choice was cultured fibroblasts (Horellou et al., 1990a,b; Uchida et al., 1992; Wolff et al., 1989).
Dopamine replacement for Parkinson’s disease (PD) have seen three major iterations of improvements since the introduction of l-3,4-dihydroxyphenylalanine (l-DOPA) pharmacotherapy: dopamine receptor agonists, ex vivo gene transfer for cell transplantation and most recently in vivo gene therapy. In this chapter, we describe the principles behind viral vector-mediated enzyme replacement in PD. We focus on the enzymes involved in the dopamine synthesis and their internal regulation, the early experimental work on gene therapy using different viral vector types and selection of transgenes, and finally discuss the recently completed early phase clinical trials in PD patients
Increased transcription of the tyrosine hydroxylase gene in individual locus coeruleus neurons following footshock stress
2000, Neuroscience
Footshock-evoked change in transcriptional activity of tyrosine hydroxylase in neurons of the locus coeruleus was examined using an intron-specific in situ hybridization histochemical technique. A significant increase in the cellular concentration of tyrosine hydroxylase primary transcripts was found in locus coeruleus neurons 3 h following 30 min of intermittent footshock. However, the footshock-induced increase in tyrosine hydroxylase transcription was not homogeneously expressed in locus coeruleus neurons. Similarly, administration of the α₂-adrenergic receptor antagonist idazoxan produced a significant increase in the cellular concentration of tyrosine hydroxylase primary transcripts that was heterogeneously distributed among locus coeruleus neurons. Both footshock and idazoxan significantly increased the regional levels of tyrosine hydroxylase messenger RNA in the locus coeruleus. The time-course of changes in tyrosine hydroxylase transcription rate and messenger RNA levels in the locus coeruleus was examined after a 15 min exposure to footshock. A robust increase in tyrosine hydroxylase transcription rate was found at the end of 15 min of footshock, which remained elevated for 6 h and was back to the control levels by 24 h. In contrast, in response to a 15 min period of footshock tyrosine hydroxylase messenger RNA concentrations in the locus coeruleus did not increase until 6 h and remained elevated at 24 h.
These findings demonstrate that transcription of the tyrosine hydroxylase gene in locus coeruleus neurons in response to footshock stress occurs rapidly, is sustained for many hours and is heterogeneously distributed. These data also suggest that the increase in tyrosine hydroxylase messenger RNA following footshock is mediated, at least in part, by an increase in tyrosine hydroxylase gene transcription.
Analysis of tyrosine hydroxylase gene transcription using an intron specific probe
2000, Journal of Neuroscience Methods
The nuclear run-on assay is the most commonly used technique to determine transcription rates of specific genes such as tyrosine hydroxylase. Its application to studies in the nervous system is problematic, however, as a result of limitations in sensitivity and the loss of anatomical integrity. We observed that the relative levels of tyrosine hydroxylase intron 2-containing RNA using a ribonuclease protection assay in the adrenal medulla changed in response to pharmacological treatments consistently with changes shown by the nuclear run-on assay. Our results indicate that measures of tyrosine hydroxylase primary transcript levels offer an alternative to the nuclear run-on assay and validate the application of intron-specific in situ hybridization as a means of assessing the relative transcriptional activity of the tyrosine hydroxylase gene. Similar quantitative results were obtained using intron-specific in situ hybridization with oligonucleotide probes specific for rat tyrosine hydroxylase intron 2. Furthermore, we observed that intron-specific in situ hybridization could be used to measure tyrosine hydroxylase transcription rates in the locus coeruleus, providing resolution at the level of single neurons. Thus, measuring the levels of tyrosine hydroxylase intron 2 provides a sensitive measure of tyrosine hydroxylase transcription rate that can be applied to the study of brain catecholaminergic neurons.
4.5 kb of the rat tyrosine hydroxylase 5' flanking sequence directs tissue specific expression during development and contains consensus sites for multiple transcription factors
1999, Molecular Brain Research
To delineate DNA sequences responsible for developmentally correct expression of the rat tyrosine hydroxylase (TH) gene, we analyzed a line of transgenic mice expressing high levels of human placental alkaline phosphatase (AP) under control of 4.5 kb of 5′ flanking DNA from the rat TH gene in embryos and adults. Several regions, such as the accessory olfactory bulb, which were not thought to synthesize TH protein or do so only transiently, were shown to express TH protein using an improved method of antigen retrieval for TH immunohistochemistry. Many of these regions had been shown to express TH-driven reporter genes in transgenic mice. In the central nervous system, AP was detected in essentially all TH-expressing cell groups throughout development and in adults. In the peripheral nervous system, transgene expression paralleled endogenous TH expression in the developing adrenal medulla and sympathetic ganglia but not in transiently TH-positive cells in dorsal root ganglia. Peripheral expression in the adult adrenal medulla was very weak and absent in sympathetic ganglia. The specificity with which the 4.5 kb region directs transgene expression in embryos is comparable to that observed with longer 5′ flanking promoter regions, implying that this region contains the control elements for appropriate expression during development. Sequence analysis of the region demonstrates a GT dinucleotide repeat, an element that resembles the neural restrictive silencer element (NRSE), which restricts transcription of neuronal genes in non-neuronal cells, and consensus sites for three families of transcription factors, Ptx1/3, Nurr1 and Gli1/2, which are required for the early differentiation of mesencephalic neurons.

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Characterization of rat and human tyrosine hydroxylase genes: Functional expression of both promoters in neuronal and non-neuronal cell types

Gene Anal. Tech.

Cell

Cell

Cell

Cell

Progress in Catecholamine Research

Mol. Cell. Biol.

Biochemistry

Biochemistry

Nature