Genome-wide screening of dioxin-responsive genes

二恶英通过芳烃受体改变下游靶基因的表达水平，影响发育分化。然而，二恶英反应基因的光谱尚未被描绘出来。本研究的目的是利用两种互补的方法：生物信息学分析和微阵列分析，以全面的方式定义二恶英反应基因的光谱。首先，我们以半全基因组的方式在假定的启动子区域（ppr）内绘制了异种生物响应元件（XREs）。XREs是二恶英诱导的几种基因的ppr中普遍存在的转录调控序列“gcgtg”。通过比对医学研究所人类全长cDNA数据库中的8060个cDNA序列，获得了2773个基因转录起始位点（TSS）上游10kb的序列，其中2843个基因在TSS上游10kb范围内具有至少3个XRE一致性。在2773个基因中，PPR内的XREs总数为26787个。来自TSS的26,787个XREs的位置绘制在图2a上。XRE倾向于出现在靠近TSS的地方。在每10kb上游序列内，所有XRE粉尘中最小粉尘的位置倾向于出现在TSS附近。小鼠分析：将FANTOM2小鼠全长cDNA数据库中的60770条cDNA序列与小鼠基因组序列比对，获得18541个基因tss上游10kb的序列。在18541个基因中，9762个基因在TSS上游10kb范围内具有至少3个XRE一致序列。在9762个基因中，PPR内的XREs总数为74725个。同样，在小鼠中，XRE倾向于出现在靠近TSS的地方。在每10kb上游序列内，所有XRE集群中最小集群的位置倾向于出现在TSS附近。其次，我们评估了暴露于原型二恶英2,3,7,8-四氯二苯并-对二恶英（TCDD）的小鼠胚胎脑的表达谱，并与未暴露的脑的表达谱进行了比较。6个杂交体（实验组3个，对照组3个）的表达值进行背景相减和归一化处理。我们使用稳健的数据驱动方法推断在TCDD暴露下差异表达的基因。约0.37%（46/12488）的评估基因在基因表达上出现显著（p<0.0001）变化，或增加或减少（表1）。40个基因中，22个基因在tcdd暴露的幼犬大脑中表达增加，24个基因表达减少。Cyp1b1和Cyp1a1，这两种基因先前都被证明是由体外暴露二恶英诱导的，在清单中被诱导。将表达水平的变化与平均强度进行对比，并通过基因检测确定bonferroni型基因的临界区间。暴露在二恶英下下调的一些基因是致病基因：Mid1、Pitx2和Gata3。这些基因的突变分别导致Opitz综合征、Rieger综合征和HDR综合征。已知人类MID1基因突变可引起腭裂，这是小鼠产前二恶英暴露的主要特征。此外，暴露组Glyoxalase 1 （GLO）表达下调。GLO1酶活性的降低已被证明会导致甲基乙二醛的积累，这可能对发育中的大脑有害。此外，一项关联研究显示，乙二醛酶I的单核苷酸多态性是自闭症的易感性因素（Junaid et al., 2004）。鉴于外源毒素在自闭症谱系发展中的潜在作用（Edelson和Cantor， 1998），暴露组GLO1减少的生物学相关性需要进一步探索。少

Dioxins alter the expression levels of downstream target genes and affect development and differentiation through the aryl hydrocarbon receptor. However, the spectrum of the dioxin-responsive genes is yet to be delineated. The purpose of the present study is to define the spectrum of dioxin-responsive genes in a comprehensive manner using two complementary approaches : bioinformatic analysis and micro-array analysis.First, we mapped the xenobiotics responsive elements[XREs] within the putative promoter regions[PPRs] in a semi genome-wide manner. XREs are the consensus transcriptional regulation sequence "gcgtg" which are present in the PPRs of several genes induced by dioxins. Human analysis Sequences 10kb upstream from the transcription start sites[TSSs] of 2,773 genes were obtained by aligning 8,060 cDNA sequences in the human full-length cDNA database of The Institute of Medical Science with the human genome sequences 2,843 out of 2,773 genes possessed at least three XRE consensus s … More equences(gcgtg) within 10kb upstream of the TSS. Total number of XREs within the PPR was 26,787 among the 2,773 genes. The position of the 26,787 XREs from the TSS was ploteted on Fig. 2a. The XRE tended to be present closer to the TSS. The position of the smallest duster among all the XRE dusters within each 10kb upstream sequence tended to be present near the TSS. Mouse analysis : Sequences 10kb upstream from the TSSs of 18,541 genes were obtained by aligning 60,770 cDNA sequences in the FANTOM2 mouse full-length cDNA database of with the mouse genome sequences. 9,762 out of 18,541 genes possessed at least three XRE consensus sequences within 10kb upstream of the TSS. Total number of XREs within the PPR was 74,725 among the 9,762 genes. Again, in mice, the XRE tended to be present closer to the TSS. The position of the smallest cluster among all the XRE clusters within each 10kb upstream sequence trended to be present near the TSS.Second, we evaluated the expression profile of the mouse embryonic brain exposed to the prototypic dioxin, 2,3,7,8-tetrarchlorodibenzo-p-dioxin (TCDD), and compared with the expression profile of the unexposed brain. The expression values for the 6 hybridizations(3 from experimental group and 3 from the control group) were background-subtracted and normalized. We inferred genes that differentially expressed under TCDD exposure using a robust data-driven method. About 0.37%(46/12488) of the assessed genes showed significant(p<0.0001) change, either increase or decrease, in gene expression(Table 1). Among the 40 genes, 22 genes increased and 24 genes decreased their expression in TCDD-exposed pup brain. Cyp1b1 and Cyp1a1, both of which have been shown previously to be induced by dioxin exposure in vitro, were induced in the list. The changes in the expression level were plotted against the average intensity and the Bonferroni-style gene by gene tests were performed to determine the critical interval. Some of genes down-regulated by dioxin exposure are disease-causing genes : Mid1, Pitx2, and Gata3. Mutations in those genes lead to Opitz syndrome, Rieger syndrome, and HDR syndrome, respectively. Mutation in human MID1 gene is known to cause cleft palate, a cardinal feature of prenatal dioxin exposure in mice. In addition, Glyoxalase 1 (GLO) was down-regulated in the exposed group. A reduction in GLO1 enzyme activity has been demonstrated to result in the accumulation of methylglyoxal, which may be toxic to the developing brain Furthermore, an association study revealed that single nucleotide polymorphism in glyoxalase I as autism susceptibility factor (Junaid et al., 2004). In view of a potential role of xenobiotic toxins in the development of autistic spectrum (Edelson and Cantor, 1998), the biological relevance of GLO1 reduction in the exposed group needs to be further explored. Less