Abstract
Nurr1 is an orphan nuclear receptor best known for its essential role in the development and maintenance of midbrain dopaminergic (DA) neurons. During DA neurogenesis, Nurr1 directly targets human tyrosine hydroxylase (hTH). Here we investigated this targeting to identify the molecular mechanisms by which Nurr1 regulates DA neurogenesis. We previously cloned the hTH promoter and found three consensus elements for Nurr1 binding: NBRE-A, -B, and -C. In the present study, gel retardation and luciferase assays using hTH constructs showed that Nurr1 preferentially bound to NBRE-A, through which it mediated transcriptional activity. Furthermore, Nurr1 displayed dual-function transcriptional activities depending on the cell type. In DA-like SH-SY5Y cells, Nurr1 dose-dependently stimulated hTH-3174 promoter activity by 7- to 11-fold. However, in the human neural stem cell (hNSC) line HB1.F3, Nurr1 strongly repressed transcription from the same promoter. This repression was relieved by mutation of only the NBRE-A element and by nicotinamide [an inhibitor of class III histone deacetylases (HDACs), such as SIRT1], but not by trichostatin A (an inhibitor of class I and II HDACs). SIRT1 was strongly expressed in the nucleus of HB1.F3 cells, while it was localized in the cytoplasm in SH-SY5Y cells. ChIP assays of HB1.F3 cells showed that Nurr1 overexpression significantly increased the SIRT1 occupancy of the NBRE-A hTH promoter region, while low SIRT1 levels were observed in control cells. In contrast, no significant SIRT1 recruitment was observed in SH-SY5Y cells. These results indicate that differential SIRT1 localization may be involved in hTH gene regulation. Overall, our findings suggest that Nurr1 exists in dual transcriptional complexes, including co-repressor complexes that can be remodeled to become co-activators and can fine-tune hTH gene transcription during human DA neurogenesis.
Highlights
The dopaminergic neurons of the midbrain dopaminergic system have been studied extensively in relation to Parkinson’s disease, and many studies have explored the possibility of using cell replacement therapy with stem cells in future treatments [1,2,3,4]
HB1.F3 cells were strongly positive for several markers, including the early CNS markers nestin and ATP-binding cassette sub-family G member 2 (ABCG2), but were negative for the mesencephalic marker Wnt1, the astrocyte marker glial fibrillary acidic protein (GFAP), and the terminally differentiated neuronal marker neurofilament heavy polypeptide (NF-H) (Figure 1 B–C)
Because transcriptional repressors often recruit histone deacetylases (HDACs) to transcriptional complexes, we evaluated whether the inhibition of human tyrosine hydroxylase (hTH) promoter activity by Nurr1 requires HDAC activity (Figure 6)
Summary
The dopaminergic neurons of the midbrain dopaminergic (mdDA) system have been studied extensively in relation to Parkinson’s disease, and many studies have explored the possibility of using cell replacement therapy with stem cells in future treatments [1,2,3,4]. In order to engineer stem cells with mdDA characteristics, the appropriate dopaminergic phenotype needs to be obtained through molecular coding [5,6,7,8]. Nurr overexpression in stem cells may be important for efforts establishing cell replacement therapies in Parkinson’s disease [12,13,14]. Despite intense interest in understanding the development of DA cells, Nurr regulation of genes important in DA neuron development has been rarely investigated
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