p300/HDAC介导的可逆的乙酰化修饰参与Th1细胞因子IL-12和IL-18基因转录水平的调控
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摘要
细胞因子Interleukin 12(IL-12)在抗原提呈细胞中的调控表达在病原体感染和炎症疾病中至关重要，Interleukin 18(IL-18)是调节先天性免疫和获得性免疫的重要细胞因子。IL-12和IL-18在Th1的发育中起着重要作用。阐明IL-12和IL-18基因表达调控机制对于治疗IL-12和IL-18相关疾病至关重要。
由组蛋白乙酰转移酶(HATs)和组蛋白去乙酰化酶(HDACs)催化的乙酰化反应在基因的转录调节中起着重要作用，这两种酶通过对核心组蛋白进行可逆修饰来调节核心组蛋白的乙酰化水平，从而调控转录的起始，并由此介导基因的激活或沉默。p300是一种重要的组蛋白乙酰化酶，在转录调控中起着重要作用，它参与了许多基因的表达调控。组蛋白去乙酰化酶(HDACs)同组蛋白乙酰转移酶(HATs)共同作用使乙酰化状态达到平衡。组蛋白的高乙酰化通常与基因活化有关，而组蛋白的低乙酰化往往与基因活性的抑制密切相关。
尽管IL-12／IL-18作为两个重要的Th1细胞因子在免疫中的重要性越来越受到重视，尽管乙酰化修饰在基因转录中的重要地位早已众所周知，但是乙酰化是否参与IL-12／IL-18的基因表达调控至今为止未见报道。本文旨在证实是否p300／HDACs介导的可逆的乙酰化作用参与了IL-12／IL-18的转录调控，探讨乙酰化调控IL-12／IL-18的机制，为IL-12 p40／IL-18相关疾病的研究奠定理论基础。
本文通过一系列共转染实验证实，p300／HDACs参与了IL-12p40／IL-18 p1启动子荧光素酶报告基因表达调控。野生型p300wt增强IL-12 p40／IL-18 p1启动子荧光素酶报告基因的活性，而HAT区缺失突变体p300△HAT不具有同样的激活效果。野生型E1A 12Swt能够抑制这种激活作用。这些结果证明p300的乙酰转移酶活性在调控TL-12 p40／IL-18 p1启动子荧光素酶报告基因的过程中是必需的。同时，我们的实验结果表明p300与IL-12 p40／IL-18 p1的相关转录因子(Ets、c-Rel、C／EBP、Spl和c-Fos)有协同作用，而转录因子与
p300么郎T没有协同作用，EIA 12Swt能够抑制这种协同作用，证明
p300叫乙酞转移酶活性对于协同作用是必需的。多种HDAC能够抑制
p300与转录因子对工L一1 2 p4o/IL一18 pl启动子荧光素酶报告基因的
协同作相。RT一PCR实验证实p3OO对于内源工L一12 p40/IL，18基因mRNA
的丰度有增强作用。染色质免疫沉淀(chIP)实验证实p30o能够提高
内源IL二12 p40启动子处的组蛋白乙酞化水平。
通过以上研究，阐明可逆的乙酞化在IL一12 p40/IL一18基因调控
中的作角，探讨乙酞化调控工L一1:p40/IL一18基因的作用机制，为IL一12
p40/工L十18相关疾病的研究奠定理论基础。
Interleukin-12 (IL-12) is a heterodimeric cytokine produced by macrophages in response to intracellular pathogens, and provides an obligatory signal for the differentiation of T-helper-1 cells. Interleukin-18 (IL-18) is a pleiotropic cytokine involved in the development of T helper type 1 (Thl) cells, and it plays important roles in regulation of both the innate and acquired immune responses.
It is now clear that the reversible acetylation/deacetylation modification of core histone tails is involved in the modulation of chromatin structure and function that leads to the activation/suppression of gene expression. This reversible modification is accomplished by two categories of enzymes, i.e., histone acetyltransferases (HATs) and histone deacetylases (HDACs). p300 is an important HAT and has implicated in the regulation of gene expression, including many cytokine genes. HDACs play the opposite role in the gene regulation. HDACs remove acetyl moieties from specific lysine residues of core histones to keep the levels of histone acetylation at a steady state.
Although it has long been known that acetylation modification plays an important role in gene transcription, whether it is involved in the regulation of IL-12/IL-18 expression had not been investigated up to this study. The aim of this study was to elucidate whether the reversible histone acetylation/deacetylation modification mediated by p300/HDACs participates in the regulation of IL-12/IL-18 transcription expression and whether the HAT activity of p300 is necessary for its function.
In this study, we analyzed the roles of p300/HDACs in the regulation of IL-12/IL-18 by using RT-PCR, ChIP and a series of co-transfection studies. The results demonstrate that p300 stimulated the activation of IL-12 p40/IL-18 pi promoter and the HAT activity of p300 was essential to its function. In addition, p300 was shown to be able to work synergistically with the transcription factors on activation of IL-12 p40/IL-18 p1 promoter and this effect could be impaired by HDACs.
Furtherajiore, p300 promoted the endogenous IL-12 p40/IL-18 mRNA synthesis and enhanced the acetylation of the histone H3 at IL-12 p40 promotejr. Results presented in this paper indicate that the reversible histone cetylation/deacetylation modification plays an important role in the trans|criptional regulation of IL-12 p40/IL-18. All these original results will be (helpful in establishing theoretical bases for the cure of diseases associated with IL-12/IL-18.
由组蛋白乙酰转移酶(HATs)和组蛋白去乙酰化酶(HDACs)催化的乙酰化反应在基因的转录调节中起着重要作用，这两种酶通过对核心组蛋白进行可逆修饰来调节核心组蛋白的乙酰化水平，从而调控转录的起始，并由此介导基因的激活或沉默。p300是一种重要的组蛋白乙酰化酶，在转录调控中起着重要作用，它参与了许多基因的表达调控。组蛋白去乙酰化酶(HDACs)同组蛋白乙酰转移酶(HATs)共同作用使乙酰化状态达到平衡。组蛋白的高乙酰化通常与基因活化有关，而组蛋白的低乙酰化往往与基因活性的抑制密切相关。
尽管IL-12／IL-18作为两个重要的Th1细胞因子在免疫中的重要性越来越受到重视，尽管乙酰化修饰在基因转录中的重要地位早已众所周知，但是乙酰化是否参与IL-12／IL-18的基因表达调控至今为止未见报道。本文旨在证实是否p300／HDACs介导的可逆的乙酰化作用参与了IL-12／IL-18的转录调控，探讨乙酰化调控IL-12／IL-18的机制，为IL-12 p40／IL-18相关疾病的研究奠定理论基础。
本文通过一系列共转染实验证实，p300／HDACs参与了IL-12p40／IL-18 p1启动子荧光素酶报告基因表达调控。野生型p300wt增强IL-12 p40／IL-18 p1启动子荧光素酶报告基因的活性，而HAT区缺失突变体p300△HAT不具有同样的激活效果。野生型E1A 12Swt能够抑制这种激活作用。这些结果证明p300的乙酰转移酶活性在调控TL-12 p40／IL-18 p1启动子荧光素酶报告基因的过程中是必需的。同时，我们的实验结果表明p300与IL-12 p40／IL-18 p1的相关转录因子(Ets、c-Rel、C／EBP、Spl和c-Fos)有协同作用，而转录因子与
p300么郎T没有协同作用，EIA 12Swt能够抑制这种协同作用，证明
p300叫乙酞转移酶活性对于协同作用是必需的。多种HDAC能够抑制
p300与转录因子对工L一1 2 p4o/IL一18 pl启动子荧光素酶报告基因的
协同作相。RT一PCR实验证实p3OO对于内源工L一12 p40/IL，18基因mRNA
的丰度有增强作用。染色质免疫沉淀(chIP)实验证实p30o能够提高
内源IL二12 p40启动子处的组蛋白乙酞化水平。
通过以上研究，阐明可逆的乙酞化在IL一12 p40/IL一18基因调控
中的作角，探讨乙酞化调控工L一1:p40/IL一18基因的作用机制，为IL一12
p40/工L十18相关疾病的研究奠定理论基础。
Interleukin-12 (IL-12) is a heterodimeric cytokine produced by macrophages in response to intracellular pathogens, and provides an obligatory signal for the differentiation of T-helper-1 cells. Interleukin-18 (IL-18) is a pleiotropic cytokine involved in the development of T helper type 1 (Thl) cells, and it plays important roles in regulation of both the innate and acquired immune responses.
It is now clear that the reversible acetylation/deacetylation modification of core histone tails is involved in the modulation of chromatin structure and function that leads to the activation/suppression of gene expression. This reversible modification is accomplished by two categories of enzymes, i.e., histone acetyltransferases (HATs) and histone deacetylases (HDACs). p300 is an important HAT and has implicated in the regulation of gene expression, including many cytokine genes. HDACs play the opposite role in the gene regulation. HDACs remove acetyl moieties from specific lysine residues of core histones to keep the levels of histone acetylation at a steady state.
Although it has long been known that acetylation modification plays an important role in gene transcription, whether it is involved in the regulation of IL-12/IL-18 expression had not been investigated up to this study. The aim of this study was to elucidate whether the reversible histone acetylation/deacetylation modification mediated by p300/HDACs participates in the regulation of IL-12/IL-18 transcription expression and whether the HAT activity of p300 is necessary for its function.
In this study, we analyzed the roles of p300/HDACs in the regulation of IL-12/IL-18 by using RT-PCR, ChIP and a series of co-transfection studies. The results demonstrate that p300 stimulated the activation of IL-12 p40/IL-18 pi promoter and the HAT activity of p300 was essential to its function. In addition, p300 was shown to be able to work synergistically with the transcription factors on activation of IL-12 p40/IL-18 p1 promoter and this effect could be impaired by HDACs.
Furtherajiore, p300 promoted the endogenous IL-12 p40/IL-18 mRNA synthesis and enhanced the acetylation of the histone H3 at IL-12 p40 promotejr. Results presented in this paper indicate that the reversible histone cetylation/deacetylation modification plays an important role in the trans|criptional regulation of IL-12 p40/IL-18. All these original results will be (helpful in establishing theoretical bases for the cure of diseases associated with IL-12/IL-18.
引文
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