Comparative Genomic Responses to Environmental Stressors

对环境压力源的比较基因组反应

• 批准号: 7968212
• 负责人: JONATHAN H FREEDMAN
• 金额: $ 140.23万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968212
• 关键词: Address; Affect; Attention; Attenuated; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; Biology; Cadmium; Caenorhabditis elegans; Calcium; Categories; Cell Nucleus; Cells; Chemicals; Code; Collaborations; Collection; Complex; Conditioned Culture Media; Congenital Abnormality; Consensus; Copper; Cultured Cells; Cytoplasmic Granules; DNA Binding; Data; Data Analyses; Development; Disease; Elements; Etiology; Exposure to; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Genomics; Head; Indium; Individual; Intestines; Lead; Longevity; Mammals; Mass Spectrum Analysis; Measures; Mercuric chloride; Mercury; Metabolic; Metabolism; Metal exposure; Metallothionein; Metals; Microarray Analysis; Modeling; Molecular; Mutation; Nature; Neurons; Nuclear; Nuclear Structure; Nuclear Translocation; Nucleic Acid Regulatory Sequences; Ontology; Organism; Pathology; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Site; Principal Component Analysis; Process; Proteins; Publications; RNA Interference; Reporter; Reporting; Resistance; Role; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Pathway Gene; Site; Site-Directed Mutagenesis; Stress; Subfamily lentivirinae; System; Tail; Temperature; Toxic Environmental Substances; Toxicant exposure; Transcriptional Activation; Transcriptional Regulation; Transition Elements; Translating; Tretinoin; Virus; Vulva; Work; Yeasts; Zinc; Zinc Fingers; biological adaptation to stress; casein kinase II; cellular transduction; comparative; environmental stressor; gain of function; human disease; interest; knock-down; loss of function; metal poisoning; mutant; novel; nrf1 protein; programs; promoter; repaired; response; second messenger; sensor; toxicant; transcription factor; transcription factor MTF-1

The overall program in the Comparative Genomics Group involves understanding contribution of environmental toxicants to the etiology of human diseases. We are currently focusing on how metal-responsive regulatory processes control the gene expression. The projects on-going in this group can be defined in two major categories: Determining the mechanism by-which the metal responsive transcription factor MTF-1 actives gene expression; and global genomic responses to metal exposure in multiple species. METAL RESPONSIVE TRANSCRIPTION FACTOR, MTF-1 Targeted site-directed mutagenesis of MTF-1- Prosite analysis predicts six PKC consensus phosphorylation sites in MTF-1. We generated a collection of site-directed MTF-1 mutants in-which combinations of five of the six the potential PKC phosphorylated residues have been modified (e.g., single, double, triple, etc. mutants). In collaboration with the lentivirus expression core a collection of virus strains containing: wild-type MTF-1, five individual PKC mutants, and a strain expressing MTF-1 containing all five PKC mutations have been produced. After transducing dko7 cells (MTF-1 nulls) with each strain of virus, the ability of cadmium and zinc to activate transcription was measured. Two mutations, T224A and S641A, were unable to activate metal-inducible transcription. The other mutations activated transcription to the level observed in dko7 cells transduced with wild-type MTF-1. Interestingly,a mutant in which all six PKC site were modified had wild-type activity. We have shown that cells expressing mutant MTF-1s are sensitive to metals and that these mutants can bind to MRE's with wildtype activity. We are currently examining their ability to translocate into the nucleus. We have also begun to generate a series of MTF-1 mutants with modified casein kinase 2 sites. GENOMIC RESPONSES TO TRANSITION METAL EXPOSURE 1. Response of retinoic acid metabolism to cadmium exposure - In the analysis of the genomic responses of C. elegans exposed to cadmium, we observed that the expression of genes encoding proteins involved in retinoic acid (RA) metabolism was affected. We translated the information from C. elegans to a mammalian system, hepa 1-6 cells, and found that exposure to cadmium increased the level of RA-responsive transcription. It also affected the steady-state levels of mRNAs encoding proteins that are involved in the synthesis and degradation of RA. Finally, preliminary results indicated that exposure to cadmium causes a significant increase the level of RA in hepa 1-6 cells. These results suggest a mechanism by-which cadmium causes birth defects. 2. Characterization of a novel metal-responsive gene from C. elegans - From the C. elegans microarray analysis we identified a novel cadmium-inducible gene, designated numr-1 (nuclear localized metal responsive). Subsequent genomic analysis identified a second gene numr-2 that is 99% identical, in both coding and regulatory regions, to numr-1. Both numrs are metal responsive and expressed in identical cells: constitutively in a sub-set of neurons in the head, vulva and tail; and in intestinal and pharyngeal cells following metal exposure. In addition, both NUMRs are targeted to punctate nuclear structures putatively identified as nuclear stress granules. Over expression of NUMR-1 caused increased resistance to metal toxicity and life-span. Likewise, knocking down NUMR-1/2 expression increased C. elegans sensitivity to metal exposure. We observed that calcium activated numr-1 transcription. This has lead to a reexamination of the relation between calcium and cadmium in transcription regulation. Much of the previous work suggesting that cadmium behaves as a calcium mimic was preformed at supra-toxicological metal concentrations (>LC70). We are examining the calcium/cadmium relation in cultured cells at metal concentrations that induce gene expression but are minimally toxic to the cell. Initial results suggests that calcium does not function as a second messenger for cadmium, until cells have begun to die. 3. Genomic responses to copper in HepG2 cells - We previously showed that copper was able to affect the activities of specific transcription factors, MTF-1, and AP-1, and their cognate signal transduction pathways. We have completed an analysis of transcriptome of HepG2 cells exposed to copper. Bioinformatic analysis of this data including Gene Ontology, Interactomes and Ingenuity has identified several novel transcription factors and signaling pathways responsible of copper-responsive gene expression. This work has been submitted for publication in Genome Biology. We examined the relation between copper exposure and the activity of the transcription factors NF-B, HNF4 and NRF-1 using RNA interference and biochemical assays. 4. Genetic screens to identify regulators of metal-inducible transcription in C. elegans We have initiated a new project to identify regulators of metal-inducible transcription using a classic genetics approach. We created mtl-1::GFP, mtl-2::GFP, numr-1::GFP and cdr-1::GFP C. elegans reporter strains to screen for metal-responsive regulators and signaling pathways. We are currently mutagenizing these strains, and will isolate gain-of-function (high levels of transcription in the absence of cadmium) and loss-of-function (no or attenuated transcription in the presence of cadmium) mutants. Ultimately, the C. elegans gene(s) and cognate pathways responsible for the phenotypes will be identified, and the role of the homologous mammalian gene(s) in regulating metal-inducible transcription will be explored. 5. Affects of mercurials on the C. elegans transcriptome - We are investigating the effects of inorganic mercury (HgCl2) and methyl mercury on gene expression in C. elegans. A direct comparison between these two mercurials at equi-toxic concentrations, under identical exposure conditions (media, temperature, etc) in a single species has not been reported. This study will address the proposal that methyl mercury and inorganic mercury affect similar genes and signaling pathways. We have generated microarray data for each mercurial at three concentrations. Initial analysis indicates that the chemicals have few genes in common. Clustering and principal components analyses clearly differentiate between the two mercurials. These results contradict the hypothesis that the biological effects of methyl mercury are caused by ionic mercury, which is released during the metabolic degradation of organic mercury. This expression data will be compared to our previous results including the C. elegans-cadmium and the yeast-mercury transcriptomes

比较基因组学组的总体计划涉及了解环境毒物对人类疾病病因学的贡献。 我们目前的重点是金属响应调控过程如何控制基因表达。 该小组正在进行的项目可分为两大类：确定金属响应转录因子 MTF-1 激活基因表达的机制；以及多个物种对金属暴露的全球基因组反应。 金属反应转录因子，MTF-1 MTF-1 的定向定点诱变-Prosite 分析预测 MTF-1 中的六个 PKC 共有磷酸化位点。 我们生成了一系列定点 MTF-1 突变体，其中六个潜在 PKC 磷酸化残基中的五个的组合已被修饰（例如，单、双、三等突变体）。 与慢病毒表达核心合作，产生了一系列病毒株，其中包含：野生型 MTF-1、五个单独的 PKC 突变体以及表达包含所有五个 PKC 突变的 MTF-1 的病毒株。 用每种病毒株转导 dko7 细胞（MTF-1 无效）后，测量了镉和锌激活转录的能力。 T224A 和 S641A 两种突变无法激活金属诱导转录。 其他突变将转录激活至野生型 MTF-1 转导的 dko7 细胞中观察到的水平。 有趣的是，所有六个 PKC 位点均被修饰的突变体具有野生型活性。 我们已经证明，表达突变体 MTF-1 的细胞对金属敏感，并且这些突变体可以与具有野生型活性的 MRE 结合。 我们目前正在检查它们转入细胞核的能力。 我们还开始生成一系列带有修饰酪蛋白激酶 2 位点的 MTF-1 突变体。 对过渡金属暴露的基因组反应 1. 视黄酸代谢对镉暴露的反应 - 在分析接触镉的线虫的基因组反应时，我们观察到编码参与视黄酸（RA）代谢的蛋白质的基因表达受到影响。 我们将来自线虫的信息转化为哺乳动物系统 hepa 1-6 细胞，发现接触镉会增加 RA 响应转录的水平。 它还影响编码参与 RA 合成和降解的蛋白质的 mRNA 的稳态水平。 最后，初步结果表明，接触镉会导致 hepa 1-6 细胞中 RA 水平显着增加。 这些结果表明镉导致出生缺陷的机制。 2.来自秀丽隐杆线虫的新型金属响应基因的表征-从秀丽隐杆线虫微阵列分析中，我们鉴定了一种新型镉诱导基因，命名为numr-1（核局部金属响应）。 随后的基因组分析确定了第二个基因 numr-2，其编码区和调控区与 numr-1 99% 相同。 两个数字都是金属反应性的，并在相同的细胞中表达：组成性地在头部、外阴和尾部的神经元子集中；以及金属暴露后的肠和咽细胞中。 此外，两个 NUMR 的目标都是点状核结构，这些核结构被认为是核应力颗粒。 NUMR-1 的过度表达导致对金属毒性的抵抗力和寿命的增加。 同样，敲低 NUMR-1/2 表达会增加线虫对金属暴露的敏感性。 我们观察到钙激活 numr-1 转录。 这导致人们重新审视钙和镉在转录调控中的关系。 先前的大部分工作表明镉具有钙模拟物的行为，都是在超毒理学金属浓度（> LC70）下进行的。 我们正在研究培养细胞中金属浓度下的钙/镉关系，该金属浓度可诱导基因表达，但对细胞的毒性最小。初步结果表明，在细胞开始死亡之前，钙不会充当镉的第二信使。 3. HepG2 细胞中铜的基因组反应 - 我们之前表明铜能够影响特定转录因子 MTF-1 和 AP-1 的活性及其同源信号转导途径。 我们已经完成了对暴露于铜的 HepG2 细胞的转录组分析。 对这些数据（包括 Gene Ontology、Interactomes 和 Ingenuity）的生物信息学分析确定了几种负责铜响应基因表达的新型转录因子和信号通路。 这项工作已提交在《基因组生物学》上发表。 我们利用 RNA 干扰和生化检测研究了铜暴露与转录因子 NF-β、HNF4β 和 NRF-1 活性之间的关系。 4. 遗传筛选以鉴定线虫中金属诱导转录的调节因子我们启动了一个新项目，以使用经典遗传学方法鉴定金属诱导转录的调节因子。 我们创建了 mtl-1::GFP、mtl-2::GFP、numr-1::GFP 和 cdr-1::GFP 线虫报告菌株来筛选金属响应调节因子和信号通路。 我们目前正在对这些菌株进行诱变，并将分离出功能获得（在没有镉的情况下高水平转录）和功能丧失（在镉存在下没有转录或减弱转录）突变体。 最终，将鉴定出负责表型的线虫基因和同源途径，并探索同源哺乳动物基因在调节金属诱导转录中的作用。 5. 汞对线虫转录组的影响 - 我们正在研究无机汞 (HgCl2) 和甲基汞对线虫基因表达的影响。 尚未报道在相同的暴露条件（介质、温度等）下对单一物种中这两种汞在等毒性浓度下的直接比较。 这项研究将讨论甲基汞和无机汞影响相似基因和信号通路的提议。 我们为每种汞在三个浓度下生成了微阵列数据。 初步分析表明，这些化学物质几乎没有共同的基因。 聚类和主成分分析清楚地区分了这两种变化。 这些结果与甲基汞的生物效应是由离子汞引起的假设相矛盾，离子汞是在有机汞的代谢降解过程中释放的。 该表达数据将与我们之前的结果进行比较，包括线虫-镉和酵母-汞转录组