Transcriptional regulation of the hypocretin/orexin gene by NR6A1

Abstract

The hypocretin (also known as orexin) neuropeptide system coordinates the regulation of various physiological processes. A reduction in Nr6a1 expression was observed in hypocretin neuron-ablated transgenic mice. To show that prepro-hypocretin transcription is functionally modulated by NR6A1, we performed chromatin immunoprecipitation (ChIP) analysis, double-immunostaining, a luciferase reporter assay, and an in utero electroporation study. ChIP analysis showed that endogenous NR6A1 binds to a putative NR6A1-binding site. Double-immunostaining indicated almost all hypocretin neurons were positive for NR6A1 immunoreactivity. NR6A1 overexpression in SH-SY5Y cells modulated hypocretin promoter activity, an effect that was countered by lacking a putative NR6A1-binding site. Electroporation with Nr6a1 in the foetal hypothalamus promoted hypocretin transcription as compared to GFP-electroporation. These experiments confirmed that NR6A1 works as a regulator for hypocretin transcription.

Figure 1. Schematic representation of the human prepro-hypocretin gene regulatory region, chromatin immunoprecipitation (ChIP)-PCR, and colocalisation of NR6A1 in the murine hypocretin neurons

(A) Schematic representation of the human prepro-hypocretin gene regulatory region. Two highly conserved regions—OE1 and OE2 (black arrows)—and five Alu repeats (ovals) have been previously reported [3]. The first residue of the transcription start site and the translation start site are marked as +1 and +88. The 18-bp region of the potential NR6A1 binding site, including the NurRE identified by the Genomatix Software GmbH (http://www.genomatix.de), is shown in bold and underlined. (B) The 18-bp region of the potential NR6A1 binding site. Asterisks show conserved nucleic acids between the human and the mouse. (C) ChIP-PCR analysis for endogenous NR6A1 binding to the proximal region of the prepro-hypocretin gene in SH-SY5Y cells. Two primer pairs, -611Fw/-467Rv and -633Fw/-431Rv, were used to amplify the DNA containing the putative NR6A1-binding site. A schematic representation of the primer binding sites is shown in the upper panel. The results of the ChIP-PCR analysis are shown in the lower panel. Ab: samples immunoprecipitated with the anti-GCNF antibody; NRS: with normal rabbit serum; W: water (negative control). (D–G) Murine lateral hypothalamic sections. (D) DAPI-labelled nuclei (blue). (E) Alexa-594 visualised murine NR6A1 (red). (F) Alexa-488 visualised murine hypocretin (green). (G) Merged image of (D), (E), and (F) immunofluorescence. Scale bar represents 20 μm.

Figure 2. Transcriptional activities of the prepro-hypocretin promoter and its deletion mutants, and activity of the NurRE on the TATA-like promoter sequence in SH-SY5Y cells

(A) SH-SY5Y cells were cotransfected with an NR6A1/TNT vector and reporter plasmids with/without the partial deletions shown in the schematic representation. The results are shown as the mean of at least eight independent experiments ± SE. +: p < 0.05 versus pGL3-basic with the pCMVTNT vector. *: p < 0.05 versus the same reporter plasmid with the pCMVTNT vector. (B) Transcriptional activities of the NurREx1-pTAL and the NurREx3-pTAL plasmids in SH-SY5Y cells. The results are shown as the mean of at least eight independent experiments ± standard error. +: p < 0.05 versus pTAL-luc with pCMVTNT vector. *: p < 0.05 versus the same reporter plasmid with the pCMVTNT vector.

Figure 3. The relative expression levels of hypothalamic genes after in utero electroporation

In utero electroporation was performed to evaluate the effect of NR6A1 on hypocretin transcription. Control mice were electroporated with pCAG-EGFP. The relative expression level of each gene was expressed as the relative expression levels of the gene-electroporated side divided by the relative expression levels of the control side (GFP, n = 6; NR6A1, n = 7). Changes in transcription levels were compared using Student’s t-test. *: p < 0.05 versus GFP electroporated-mice.