基于组蛋白甲基化表观遗传修饰研究芳香烃受体（AhR）介导的致癌分子毒理机制

Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays important role in normal physiology with endogenous ligands and mediates the adverse effects of several classes of exogenous ligands including PAHs and dioxins. Transcriptional co-regulators mediate gene expression through covalent histone modifications. However, involvement of histone methyltransfeases (HMTs) and demethylases (HDMs) in AhR-mediated transcription still remains unknown. Firstly, to screen co-regulator activity of HMTs/HDMs for AhR-mediated transcription, the expression vector of different HDMs including LSD1, Jmjd1a, Jmjd1b, Jmjd1c, Jmjd2b, Jmjd2c, Jmjd2d, Jarid1a, Jarid1b, Jarid1c, Jarid1d and HMTs including Mll5 or Suv39H1 together with AhR reporter vector were co-transfected into 293F cells and analyzed by Luciferase reporter assay, respectively. According to the results, we observe that LSD1 and Jmjd1a promotes the transcriptional activity of AhR as a co-activator without ligand specific. In the present study, the following plans will be adopted to elucidate the molecular mechanism of histone demethy lation mediate the transcription of AhR in cells and mouse.1) To understand whether LSD1 or Jmjd1a is able to interact with AhR in cellular environments, immunoprecipitation (IP) will be performed after transiently expressing LSD1 or Jmjd1a with FLAG-tagged AhR in 293F cell; 2) To make clear the effects of LSD1 or Jmjd1a on endogenous AhR target gene (cyp1a1 and cyp1b1) expression, the gene expression was dectected by real-time PCR when LSD1 or Jmjd1a was knockdown by shLSD1 and shJmjd1a; 3) Chromatin Immunoprecipitation (ChIP) assay will be performed to determine whether AhR, LSD1 and Jmjd1a are recruited to the promoter of target genes cyp1a1 and cyp1b1; 4) To determine what kind of demethylation (H3K9me1, me2 or me3? H3K4me or H3K4me2?) activated by LSD1 or Jmjd1a at the promoter region of AhR target genes of cyp1a1 and cyp1b1, ChIP assay are performed after knockdown of LSD1 and Jmjd1a in cells; 5) Determine the possible function of LSD1 and Jmjd1a in mouse during the tumour activation. Histone methylation and demethylation are considered as a hot point of epigenetic regulation of transcription. The results of the present study would clarify the molecular mechanism of histone demethylation mediating the transcription of AhR.

多环芳烃和二噁英通过激活芳香烃受体（AhR）介导的转录途径而诱导疾病如癌症产生。组蛋白去甲基化酶是一些受体的转录共调解因子，但在AhR介导的转录调控及致肿瘤中的作用仍未知。本研究拟通过在细胞中进行基因过表达和抑制表达确认LSD1和Jmjd1a对AhR转录活性影响；用免疫沉淀技术论证LSD1或Jmjd1a在细胞内和AhR的蛋白质互作效应；进一步用染色质免疫沉淀技术分析当AhR转录通路激活时，其靶标基因增强子和启动子区域组蛋白甲基化修饰水平的变化，并通过基因抑制LSD1和Jmjd1a表达确定它们在AhR靶标基因增强子或启动子区域去甲基化的修饰位点和状态。用动物实验进一步论证LSD1和Jmjd1a在配体激活的AhR转录通路中的作用并解析其在肿瘤发生的作用。通过该项目的实施明确组蛋白去甲基化酶在AhR介导的基因转录调控和信号转导中的功能，为解析由多环芳烃等诱导基因异常表达和癌症发生机制提供证据。

多环芳烃和二噁英通过激活芳香烃受体（AHR）介导的转录途径而诱导疾病如癌症产生。组蛋白去甲基化酶是一些受体的转录共调解因子，但在AhR介导的转录调控及致肿瘤中有重要的作用。细胞的实验证据表明，组蛋白去甲基化转移酶LSD1和Jmjd1a过表达可以显著增加AHR的转录活性，当对内源性LSD1或Jmjd1a进行表达抑制时，AHR的转录活性以及其下游靶标基因的表达量会显著的抑制。同时，在LSD1过表达和正常情况下，在AHR配体3MC存在的条件下，LSD1和AHR之间存在蛋白质互作效应，但是Jmjd1a无论在过表达还是在正常的条件下，都不能检测到与AHR存在明显的互作效应。同时用染色质免疫共沉淀技术检测发现在AHR转录激活时，LSD1被迅速有效的募集到AHR启动子和增强子区域附近，与AHR形成复合体。尽管我们检测到AHR的靶标基因激活表达时，cyp1a1和cyp1b1的启动子和增强子区域组蛋白的一些氨基酸如赖氨酸上甲基化都有显著的降低，但当在细胞内抑制LSD1表达时，这些组蛋白甲基化的状态并没有发生显著的改变，这个结果说明这些基因激活是启动子和增强子区域的去甲基化状态可能不是由LSD1唯一调控的，或则LSD1作用的靶标是组蛋白的其它氨基酸残基或则构成转录复合体的其它蛋白质的残基上。以上的研究表明组蛋白的表观遗传修饰的改变对AHR以及下游相关癌已经的表达有非常重要的调控作用。