Nurr1 Represses Tyrosine Hydroxylase Expression via SIRT1 in Human Neural Stem Cells

<div><p>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.</p></div

Transactivation of the human TH gene promoter by Nurr1.

<p>(A) Schematic of the human TH promoter deletion constructs. The locations of NBRE sites A–C, the CRE site, and TATA box are shown. The longest promoter fragment starts at –3174 bp, and all of the constructs have common 3′-ends at +145 bp with respect to the transcriptional initiation site. (B and C) Responsiveness of the TH promoters to Nurr1 in F3 (B) and SH-SY5Y cells (C). Each deletion construct of phTH3174 to phTH-B and pSV-β-gal was transfected into F3 and SH-SY5Y cells in the presence or absence of pLPCNurr1. To compare Nurr1 transactivation activities directly, the luciferase activity of each reporter construct in the presence of empty vector (pLPCX) was set to 100%. The figure represents the mean ±S.D. (bar) value of triplicate samples in a representative experiment. The experiments were repeated three times with similar results.</p

SIRT1 associates with the hTH promoter in an hNSC-specific manner.

<p>(A) Expression levels of SIRT1. SIRT1 mRNAs (left panel) and proteins (right panel) were detected by RT-PCR and immunoblotting with anti-SIRT antibody in the hNSC lines HB1.F3 and HB1.A4, and in SH-SY5Y cells. (B) Immunofluorescent images in HB1.F3, HB1.A4 (left), and SH-SY5Y cells (right), using anti-SIRT1 antibody (green). Nuclei were stained with DAPI (blue). (C) Quantification of SIRT1 localization in HB1.F3 and SH-SY5Y cells. Localization was scored as nuclear, cytoplasmic, or ubiquitous (mean±se; n = 86 for HB1.F3 and n = 117 for SH-SY5Y cells). (D) Binding of SIRT1 to the hTH promoter was evaluated by ChIP assay in HB1.F3 cells transfected with no DNA (Control), pLPCX (Vector), or pLPC-Nurr1 (Nurr1). DNA fragments covering NBRE-A on the hTH promoter are indicated in the upper panel. Data are from one representative experiment of three. **<i>P</i><0.001, Student’s <i>t</i>-test.</p

Sequence-specific binding activity of Nurr1 to human TH NBREs.

<p>(A) Schematics of the human TH promoter and NBRE oligonucleotides used in this study. The NBRE sites are underlined and mutations are shown in bold. (B) EMSA with ³²P-labeled oligonucleotides containing human NBREs using nuclear extracts from SH-SY5Y cells. A slow-migrating complex (arrowhead 1) was detected, compared to free-probe migration (arrowhead 2). This pattern was supershifted in the presence of Nurr1 antibodies (arrowhead 3). The DNA-binding activity in the presence of the three TH NBREs was competed out by adding a 40- or 80-fold molar excess of each unlabeled NBRE oligonucleotide. (C) Competition assays between the NBRE-A and NBRE-B or -C sites. Nuclear extract from SH-SY5Y cells transiently transfected with the pLPC-Nurr1 plasmid was incubated with radiolabeled NBRE-A DNA in the presence or absence of 40- or 80-fold molar excess of competitor DNA as indicated above the lanes. (D) The DNA-binding activity in the presence of NBRE-A was competed out by adding a 40- or 80-fold molar excess of unlabeled wild-type oligonucleotide (A) but not by adding mutated NBREs (M1 or M2).</p

A class III HDAC is required for Nurr1-mediated suppression of TH gene transcription.

<p>(A–D) The TH reporter construct was transiently co-transfected into F3 cells with the pLPCX (closed bar) or pLPC-Nurr1 (gray bar) plasmids, and luciferase activity was determined after 24 h of treatment with TSA (A), NaB (B), VPA (C), or NAM (D). The mean activity of cells transfected with only reporter construct was set to 100%. (E) Identification of NBRE-A as a direct target of both Nurr1 and class III HDAC. Luciferase assays were performed using F3 cells transfected with the hTH-3174 or mA luciferase reporter, with or without the indicated expression vectors (200 ng; full-length Nurr1 or parent vector), and in the presence or absence of histone deacetylase inhibitors, TSA (100 ng/ml), or nicotinamide (NAM, 10 mM). Data were normalized to values for parental cells expressing only the wild-type hTH-luciferase construct (first bar). *<i>P</i><0.05.</p

Model of the regulation of the TH gene by the SIRT1 co-repressor complex in the NBRE-A element.

<p>In neural precursor cells (top), the TH gene is maintained in a repressed state by the action of a co-repressor complex containing SIRT1 at the NBRE-A element and class I and II HDACs at the CRE and TATA elements, respectively. As hNSCs finish differentiation (bottom), DA-inductive signaling (e.g., Notch or Wnt) switches Nurr1-associated proteins from co-repressors to co-activators at the NBRE-A site. In addition, the class I and II HDACs may be down-regulated or modified to inactive forms, and RNA polymerases replaces HDACs in CRE sites. Both interactions cooperatively induce TH transcription.</p

Expression of lineage-specific markers in HB1.F3 and SH-SY5Y cells.

<p>(A) Morphology of the hNSC line HB1.F3 and the DA neuron-like SH-SY5Y cells. (B) Semi-quantitative RT-PCR analysis of the early CNS, mesencephalic, neuronal, astrocyte, and dopaminergic markers in HB1.F3 and SH-SY5Y cells. (C) Immunocytochemical analysis of the NSC marker nestin in HB1.F3 cells. (D) Western blot analysis of whole extracts from HB1.F3 and SH-SY5Y cells. Cells were transfected with pLPCX or a Nurr1 expression vector, and cell lysates were subjected to immunoblotting with anti-Nurr1 (upper panel) and β-actin (internal control).</p