Functional characterization of the neuron‐restrictive silencer element in the human tryptophan hydroxylase 2 gene expression
Functional characterization of the neuron‐restrictive silencer element in the human tryptophan hydroxylase 2 gene expression
复制标题
人色氨酸羟化酶 2 基因表达中神经元限制性沉默元件的功能特征
DOI：
10.1111/jnc.14060
复制
发表时间：
2017
影响因子：
4.7
通讯作者：
Hiroaki Matsui
中科院分区：
文献类型：
--
作者：
Yukino Nawa;H. Kaneko;M. Oda;Masaaki Tsubonoya;T. Hiroi;M. Gentile;L. Colucci;R. Takahashi;Hiroaki Matsui
色氨酸羟化酶2(TPH2)是合成神经元5-羟色胺的关键酶。尽管先前的研究表明TPH2神经元限制性沉默元件(NRSE)依赖于神经元限制性沉默因子(NRSF)的活性而发挥负性调节作用，但其潜在的机制尚未完全阐明。在这里，我们详细分析了NRSE介导的人TPH2(HTPH2)启动子活性在RN46A细胞中的抑制活性，RN46A细胞是一种来自大鼠中缝神经元的细胞系。实时定量RT-PCR分析显示，5-羟色胺能标志基因(Mash1、Nkx2.2、Gata2、GATA3、Lmx1b、Pet-1、5-Htt和VMAT2)和Nrsf基因在RN46A细胞中均有表达。TPH1是RN46A细胞中表达最普遍的形式，TPH2也有表达，但表达水平较低。电泳迁移率改变分析和报告分析表明，hTPH2 NRSE是有效结合NRSF的DNA和依赖于NRSF抑制hTPH2启动子活性所必需的。HTPH2启动子的活性可通过敲除NRSF或过表达工程NRSF(显性-阴性突变体或DNA结合结构域和激活结构域融合蛋白)而增加。研究发现，组蛋白脱乙酰酶(HDAC)I类抑制剂MS-275比组蛋白去乙酰酶II类抑制剂MC-1568更能增强hTPH2启动子活性。此外，用泛素特异的蛋白酶7去泛素酶抑制剂P-22077或HBx 41108处理，可以增加hTPH2启动子的活性。综上所述，我们的数据表明，hTPH2 NRSE通过NRSF介导的启动子抑制涉及I类HDAC，并受泛素特异性蛋白酶7介导的NRSF的脱泛素化和稳定化调节。
Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 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 TPH2 (hTPH2) promoter activity in RN46A 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 RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF‐dependent repression of the hTPH2 promoter activity. The hTPH2 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 hTPH2 promoter activity. Furthermore, treatment with the ubiquitin‐specific protease 7 deubiquitinase inhibitors, P‐22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE‐mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin‐specific protease 7‐mediated deubiquitination and stabilization of NRSF.
登录
查看更多内容
DOI：
10.1073/pnas.96.17.9873
发表时间：
1999-08-17
影响因子：
11.1
作者：
Andrés, ME;Burger, C;Mandel, G
通讯作者：
Mandel, G
影响因子：
5
作者：
Kiyasova, Vera;Bonnavion, Patricia;Fernandez, Sebastian P.
通讯作者：
Fernandez, Sebastian P.
影响因子：
5.6
作者：
Gutknecht, Lise;Kriegebaum, Claudia;Lesch, Klaus-Peter
通讯作者：
Lesch, Klaus-Peter
影响因子：
14.9
作者：
Immaneni, A;Lawinger, P;Majumder, S
通讯作者：
Majumder, S