Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: Implications for the engineering of a human TH minimal promoter assembled in a self‐inactivating lentiviral vector system

Romano, Gaetano; Suon, Sokreine; Jin, Hao; Donaldson, Angela E.; Iacovitti, Lorraine

doi:10.1002/jcp.20319

Cited by 27 publications

(52 citation statements)

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“…Profile of gene expression and specificity for human TH-positive cells of the 6.3 kb minimal promoter and 1.2 kb fragment of the first intron of the human TH gene A previous study showed that a human TH minimal promoter driving GFP termed pGR382 (−194/+35) had a considerably high transcriptional activity in hNPCs, while it was silent in human skin carcinoma A431 cell line (Romano et al, 2005). However, this construct was not specific for TH-expressing differentiated hNPCs (Romano et al, 2005).…”

Section: Resultsmentioning

confidence: 99%

“…The degree of homology between the human and mouse TH promoters is about 46.6% (determined with a Clustalx program; Romano et al, 2005), whereas the human and rat TH promoters share only a 30% degree of homology (Gandelman et al, 1990;Kim et al, 2003b). The five CRs were placed upstream of the first-194 bp from the transcription start of the human TH promoter and the first 35 bp of the untranslated messenger RNA leader of the human TH gene (Romano et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…Transduced cells were then treated in vitro with a mixture of differentiating agents to enhance TH expression (Du and Iacovitti, 1997). Interestingly, the human TH minimal promoter encoding for the five CRs exhibited a significant degree of specific gene expression only in induced TH-positive hNPCs (Romano et al, 2005), while it failed to do so in TH-positive differentiated mouse primary striatal cells and in differentiated mouse substantia nigra cells (Romano et al, 2005). This finding is consistent with differences in the mechanism of TH gene regulation between the human and mouse systems.…”

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confidence: 99%

“…However, addition of genetic elements located upstream of the region (−194/+35) reduced transcriptional activity of recombinant human TH minimal promoters, indicating that a number of repressors of transcription are present inside the promoter (Kim et al, 2003b;Romano et al, 2005). Deletion analysis found a repressor of transcription in the region (−1,232/−1,210) of the human TH promoter (Kim et al, 2003b), whereas a study on evolutionary CRs among human, mouse and rat TH promoters identified another sequence encoding for repressor(s) of transcription in the region (−2,423/−2,327), which corresponds to CR-V (Romano et al, 2005). The transcriptional activity of a 6.3 kb human TH minimal promoter encoding for the region (−6,244/+35) was also very low (Fig.…”

mentioning

confidence: 99%

“…Only five short regions are evolutionarily conserved among the human, mouse and rat TH promoters (Kessler et al, 2003). A human TH minimal promoter encoding for the five CRs placed upstream of the (−194/ +35) sequence of the human TH promoter was assembled in a self-inactivating lentiviral vector system and used to transduce GFP into hNPCs and mouse primary striatal and substantia nigra cells, which were in turn treated in vitro with a mixture of differentiating agents to enhance TH gene expression (Romano et al, 2005).Interestingly, the five CRs could achieve a significant degree of specificity for TH-expressing cells only in hNPCs and not in the mouse cell culture systems (Romano et al, 2005) ROMANO et al …”

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confidence: 99%

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Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene

Romano

Macaluso

Lucchetti

et al. 2006

Journal Cellular Physiology

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The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (−6,244/ −194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (−448/+1,204). The presence of nucleosomes was observed approximately from position (−20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons.The elucidation of the mechanism of human tyrosine hydroxylase (TH) gene regulation is one of the key issues in the field of neurology. The function of TH consists of catalyzing tyrosine hydroxylation in the synthesis of L-dopa (Nagatsu et al., 1964), which is the rate-limiting step in the generation of catecholamine neurotransmitters of the central and peripheral nervous systems (Zigmond et al., 1989). There are a number of important reasons to continue to explore the complex mechanism of TH gene regulation. For instance, aberrant TH gene expression is observed in alcoholism and in psychiatric illnesses, such as schizophrenia and bipolar disorder (Ishiguro et al., 1998). In addition, the degeneration of TH-positive dopaminergic neurons of the substantia nigra is associated with Parkinson's disease (Moore, 2003).Many studies have been conducted to characterize the factors that come into play in TH gene regulation. These studies focused mainly on the human (Kessler et al., 2003;Romano et al., 2005;Jin et al., 2006), mouse (Kim et al., 2003a) and rat models (Gandelman et al., 1990 et al., 2003b). In a previous report, a 13 kb DNA fragment containing the human TH promoter was isolated from a genomic DNA library, sequenced and utilized to generate both a transgenic animal model (Kessler et al., 2003) and a variety of recombinant minimal TH promoters assembled in a self-inactivating lentiviral vector system (Romano et al., 2005). All...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene

Romano

Macaluso

Lucchetti

et al. 2006

Journal Cellular Physiology

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Tyrosine hydroxylase gene regulation in human neuronal progenitor cells does not depend on Nurr1 as in the murine and rat systems

Jin

Romano

Marshall

et al. 2005

Journal Cellular Physiology

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A previous study on the human tyrosine hydroxylase (TH) promoter revealed remarkable differences in the mechanism of TH gene regulation between the human and murine models. Indeed, a low degree of homology was observed in the sequence of TH promoters among human, mouse, and rat systems. Only five short conserved regions (CRs) could be identified among the three species. A human TH minimal promoter was engineered and assembled into a self-inactivating lentiviral vector system. This human TH minimal promoter contained the five CRs plus the first -194 bp from the transcription start of the human TH promoter and the first 35 bp of the untranslated messenger RNA leader of the human TH gene. A significant degree of specificity for this human TH minimal promoter was observed only for human neuronal progenitor cells (hNPCs), but not for TH-positive differentiated mouse primary striatal and substantia nigra cells, indicating a significant difference in TH gene regulation between the human and mouse systems. Not only is the degree of homology between the human and mouse promoters in the range of only 46%, but also those few elements that share a high degree of homology display totally different functions in human and mouse brain-derived cells. In the rodent system, NR4A2 (Nurr1) is required for the transactivation of TH minimal promoters. Intriguingly, neither the dimeric nor the heterodimeric binding sites for Nurr1 are present in the 13 kb DNA sequence that contains the human TH promoter. Instead, the CRs termed one and four of the human TH promoter encode only for a half palindromic binding site sequence for Nurr1, which failed to bind Nurr1 in an in vitro electrophoretic mobility shift assay (EMSA). Additionally, of the three monomeric NGFI-B response element (NBRE) core sites (AGGTCA) and two NBRE-related sites present in the human TH promoter, only one core and two NBRE-related sites formed protein binding complexes. Interestingly, there was no increase of protein binding complex formation upon TH induction and in no case could antibodies supershift Nurr1 from the complex. These findings, taken together, demonstrate that NBRE-related binding sites for Nurr1 do not play a direct role in mediating an interaction between Nurr1 and the human TH promoter. Likewise, immunohistochemical and Western blot analysis have also confirmed that both endogenous and exogenous Nurr1 expression does not positively correlate with TH gene expression in hNPCs, in contrast to the mouse model. In addition, real-time PCR analysis revealed that the downregulation The regulation of tyrosine hydroxylase (TH) gene expression has attracted much attention in the field of neurology. Indeed, the biological function of this enzyme was investigated for four decades (Nagatsu et al., 1964). TH catalyzes the hydroxylation of tyrosine in the production of L-dopa (Nagatsu et al., 1964), which is the rate-limiting step in the synthesis of catecholamine neurotransmitters of the central and peripheral nervous systems (Zigmond et al., 1989). The degen...

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Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter

Choi

Yang

Park

et al. 2012

J of Neuroscience Research

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Pathogenesis of Parkinson's disease and related catecholaminergic neurological disorders is closely associated with changes in the levels of tyrosine hydroxylase (TH). Therefore, investigation of the regulation of the TH gene system should assist in understanding the pathomechanisms involved in these neurological disorders. To identify regulatory domains that direct human TH expression in the central nervous system (CNS), we generated two transgenic mouse lines in which enhanced yellow fluorescent protein (EYFP) is expressed under the control of either 3.2‐kb (hTHP‐EYFP construct) human TH promoter or 3.2‐kb promoter with 2‐kb 3′‐flanking regions (hTHP‐ex3‐EYFP construct) of the TH gene. In the adult transgenic mouse brain, the hTHP‐EYFP construct directs neuron‐specific EYFP expression in various CNS areas, such as olfactory bulb, striatum, interpeduncular nucleus, cerebral cortex, hippocampus, and particularly dentate gyrus. Although these EYFP‐positive cells were identified as mature neurons, few EYFP‐positive cells were TH‐positive neurons. On the other hand, we could detect the EYFP mRNA expression in a subset of neurons in the olfactory bulb, midbrain, and cerebellum, in which expression of endogenous TH is enriched, with hTHP‐ex3‐EYFP transgenic mice. These results indicate that the 3.2‐kb sequence upstream of the TH gene is not sufficient for proper expression and that the 2‐kb sequence from the translation start site to exon 3 is necessary for expression of EYFP in a subset of catecholaminergic neurons. © 2012 Wiley Periodicals, Inc.

show abstract

Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: Implications for the engineering of a human TH minimal promoter assembled in a self‐inactivating lentiviral vector system

Cited by 27 publications

References 40 publications

Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene

Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene

Tyrosine hydroxylase gene regulation in human neuronal progenitor cells does not depend on Nurr1 as in the murine and rat systems

Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter

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