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Functional characterization of the neuron‐restrictive silencer element in the human tryptophan hydroxylase 2 gene expression
Author(s) -
Nawa Yukino,
Kaneko Hanae,
Oda Masayuki,
Tsubonoya Masaaki,
Hiroi Tomoko,
Gentile Maria Teresa,
ColucciD'Amato Luca,
Takahashi Ryoya,
Matsui Hiroaki
Publication year - 2017
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.14060
Subject(s) - tph2 , microbiology and biotechnology , biology , promoter , tryptophan hydroxylase , psychological repression , gene knockdown , repressor , gene expression , gene , biochemistry , serotonergic , serotonin , receptor
Tryptophan hydroxylase 2 ( TPH 2) is the key enzyme in the synthesis of neuronal serotonin . Although previous studies suggest that TPH 2 neuron‐restrictive silencer element ( NRSE ) functions as a negative regulator dependent on neuron‐restrictive silencer factor ( NRSF ) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE ‐mediated repression of the human TPH 2 ( hTPH 2) promoter activity in RN 46A cells, a cell line derived from rat raphe neurons. Quantitative real‐time RT ‐ PCR analysis revealed the expression of serotonergic marker genes ( Mash1 , Nkx2.2 , Gata2 , Gata3 , Lmx1b , Pet‐1 , 5‐Htt , and Vmat2 ) and Nrsf gene in RN 46A cells. Tph1 mRNA is the prevalent form expressed in RN 46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH 2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF ‐dependent repression of the hTPH 2 promoter activity. The hTPH 2 promoter activity was increased by knockdown of NRSF , or over‐expression of the engineered NRSF (a dominant‐negative mutant or a DNA ‐binding domain and activation domain fusion protein). MS ‐275, a class I histone deacetylase ( HDAC ) inhibitor, was found to be more potent than MC ‐1568, a class II HDAC inhibitor, in enhancing the hTPH 2 promoter activity. Furthermore, treatment with the ubiquitin‐specific protease 7 deubiquitinase inhibitors, P‐22077 or HBX 41108, increased the hTPH 2 promoter activity. Collectively, our data demonstrate that the hTPH 2 NRSE ‐mediated promoter repression via NRSF involves class I HDAC s and is modulated by the ubiquitin‐specific protease 7‐mediated deubiquitination and stabilization of NRSF.