Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell‐specific manner

Abstract

Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step of catecholamine synthesis and its expression is necessary for neurotransmitter specification of all catecholaminergic neurons, while dopamine b-hydroxylase (DBH) is essential for the noradrenergic phenotype. In the present study, we show that Nurr1, an orphan nuclear receptor critical for dopaminergic (DA) neuron development, directly transactivates the promoter activity of the TH gene in a cell type-dependent manner, while it does not regulate the DBH promoter. Consistent with these results, only the TH promoter contains multiple sequence motifs homologous to the known Nurr1-binding motif, NBRE. TH promoter deletional analysis indicates that < 1.0 kb upstream sequences, encompassing three NBRE-like motifs (i.e. NL1, NL2 and NL3) are mostly responsible for the effects of Nurr1. Among these potential motifs, site-directed mutational analysis showed that NL1, residing from ) 35 to ) 28 bp, was most critical for mediating the transactivation by Nurr1. Strikingly, however, both DNase I footprinting and electrophoretic mobility shift assays showed that NL3, but not NL1 or NL2, has high binding affinity to Nurr1. To determine whether the proximity of these motifs may be important for transactivation by Nurr1 in the transient transfection assay, we generated reporter gene constructs in which NL3 is immediately proximal to the TATA box. Indeed, NL3 was more efficient in this position than NL1 or NL2 for mediating the transactivation by Nurr1. Our results suggest that Nurr1 may play a direct role for specification of DA neurotransmitter identity by activating TH gene transcription in a cell context-dependent manner.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease

“…3C). In vivo chromatin immunoprecipitation (ChIP) assay clearly showed that Nurr1 is recruited to both NL1 and NL3 sites of the TH promoter in AQ-and CQ-treated cells (20), suggesting that Nurr1 is directly involved in AQ-and CQmediated induction of TH gene expression (Fig. 3D).…”

“…In our original assays using the reporter construct p4xNL3-Luc containing four copies of the NBRE-like NL3 motif residing in the TH promoter (20), AQ and CQ activated luciferase reporter activity up to approximately 3-fold in a dose-dependent manner ( Fig. 1B and SI Appendix, Fig.…”

“…To identify small molecules that can activate Nurr1, we established efficient high-throughput assay systems based on our previous study showing that Nurr1 prominently activates the tyrosine hydroxylase (TH) gene promoter function (20). We used the reporter construct p4xNL3-Luc (containing four copies of the NGFI-B responsive element (NBRE)-like motif (NL3), identified in the TH upstream promoter positioned immediately proximal to the TATA box and linked to the luciferase gene) along with pCMV-Nurr1 construct (full-length Nurr1-expression vector) in human neuroblastoma SK-N-BE(2)C cells, followed by treatment with each compound for 24 h followed by measurement of luciferase activity.…”

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

“…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).…”

“…Another striking difference between the human and murine models was observed in a more recent study on the nuclear orphan receptor NR4A2 (Nurr1), which did not affect human TH gene expression in hNPCs, in contrast to the mouse and rat systems (Jin et al, 2006). Nurr1 is required to transactivate mouse TH minimal promoters (Iwawaki et al, 2000;Kim et al, 2003a), as well as other unrelated promoters, such as the osteocalcin promoter in mouse osteoblasts (Pirih et al, 2004). However, TH gene expression did not depend on a direct Nurr1-mediated transactivation in the human model (Jin et al, 2006).…”