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Functional Characterization of Neural-Restrictive Silencer Element in Mouse Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Gene Expression

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Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in −1,601 to −1,581 bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5′ flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE–NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene.

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Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research #21591182 (to A.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr. Yuki Kambe and Takashi Kurihara for their helpful discussion. We also acknowledge Drs. Kenji Kangawa and Akira Arimura for their support throughout the course of this work.

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Authors and Affiliations

  1. Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan

    Hideki Sugawara, Aiko Tominaga, Kazuhiko Inoue & Atsuro Miyata

  2. Department of Clinical Pharmacy and Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, 890-8520, Japan

    Hideki Sugawara, Yasuo Takeda & Katsushi Yamada

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  1. Hideki Sugawara
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  2. Aiko Tominaga
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  3. Kazuhiko Inoue
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  4. Yasuo Takeda
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  5. Katsushi Yamada
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  6. Atsuro Miyata
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Correspondence to Atsuro Miyata.

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Sugawara, H., Tominaga, A., Inoue, K. et al. Functional Characterization of Neural-Restrictive Silencer Element in Mouse Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Gene Expression. J Mol Neurosci 54, 526–534 (2014). https://doi.org/10.1007/s12031-014-0348-x

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  • DOI: https://doi.org/10.1007/s12031-014-0348-x

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