Human Genes Affecting Chromosome Metabolism and Stress Response

影响染色体代谢和应激反应的人类基因

• 批准号: 8336585
• 负责人: MICHAEL A RESNICK
• 金额: $ 161.47万
• 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/8336585
• 关键词: Address; Adjuvant Therapy; Affect; Alveolar Macrophages; Binding; Binding Sites; Biological; Blood; Cancer cell line; Cells; Cessation of life; Chemicals; Chromosomes; Clinical Research; Collaborations; Consensus; DNA; DNA Binding; DNA Damage; DNA Repair; Data; Development; Dimensions; Estrogen Receptors; Estrogens; Evaluation; Evolution; Exhibits; Exposure to; FLT1 gene; Family; Flagellin; Gene Expression; Gene Targeting; Genes; Genetic Polymorphism; Genetic Transcription; Genome; Goals; Human; Human Biology; Human Cell Line; IL8 gene; Immune response; In Vitro; Individual; Inflammatory; Inflammatory Response; Interleukin-6; Lead; Ligands; Lymphocyte; Malignant Neoplasms; Maps; Measures; Mediating; Mediator of activation protein; Messenger RNA; Metabolic; Metabolism; Modeling; Modification; Mutation; NF-kappa B; National Institute of Environmental Health Sciences; Natural Immunity; Pathway interactions; Pattern; Phenotype; Phosphorylation; Post-Translational Protein Processing; Primates; Production; Protein p53; Proteins; Receptor Gene; Receptor Signaling; Regulation; Reporter; Response Elements; Rodent; Role; Saccharomycetales; Site; Specificity; Stress; System; TLR5 gene; TLR8 gene; TP53 gene; Toll-Like Receptor 2; Toll-Like Receptor Family Gene; Toll-Like Receptor Pathway; Toll-like receptors; Transactivation; Transfection; Translating; Tumor Suppression; Tumor Suppressor Genes; Universities; Variant; Vascular Endothelial Growth Factors; Yeasts; arm; base; biological adaptation to stress; cancer cell; carcinogenesis; clinically relevant; cytokine; gene induction; genome wide association study; genome-wide; healthy volunteer; human DNA; in vivo; loss of function; macrophage; member; mitogen-activated protein kinase p38; mutant; next generation; novel; promoter; receptor expression; repaired; response; stressor; tool; transcription factor; tumor

The p53 tumor suppressor and master regulator is central to human DNA repair, damage checkpoints and many aspects of human biology. Importantly, most cancers are altered for p53 function. We have discovered a greatly expanded universe of p53 targets and human diversity as well as variations in stress responses. The DNA binding and transactivation of p53 is critical for tumor suppression. There is considerable variation in p53 dependent expression across 100s of targeted genes leading to differences in p53-mediated biological consequences. This is due in part variation in target response element (RE) sequence. We found that the target sequence motif is differs considerably from the in vitro derived RE consensus target sequence consisting of 2 copies of RRRCA/TT/AGYYY separated by a spacer of up to 13 bases. We have focused on RE functionality, i.e., the ability of REs to support transactivation by p53. To directly assess transactivation responsiveness of human REs, we developed promoter systems in budding yeast for variable p53 expression. This has been used to establish the functional evolution of REs across species and has been summarized in our piano model that describes functional variability within a transcriptional network. We have translated many of the findings human p53 in yeast to human cells in culture and ex vivo. Our previous studies showed that a SNP in the Flt1 gene promoter generates a perfect p53 -site and enables p53-mediated transactivation of FLT-1, placing the VEGF system directly in the p53 network. We found that in general p53 can provide transactivation through noncanonical binding sequences including sites. This finding greatly expands the p53 master regulatory network. We extended these studies genome-wide using ChIP-Seq analysis to identify p53 binding sites and associated gene expression changes following p53 activation by different agents in cancer cells. Interestingly, binding often is not associated with transactivation. A de novo motif search for the in vivo consensus binding sequence revealed the p53 canonical motif is, in fact, 2 tandem RRRCWWGYYY decamers without spacer. Although less efficient, p53 can bind non-canonical p53 motifs composed of either two-decamers with spacers or just a site. INTERACTION OF p53 AND ER REGULATORY NETWORKS. We had identified a 1/2-site estrogen receptor RE that greatly increased p53 transactivation at the FLT1 1/2-site p53 RE, establishing a new dimension to the p53 regulatory network. Recently, we addressed the generality of synergistic transactivation by p53 and ER. p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. The increased transactivation extends to several cancer-associated p53 mutants, suggesting ER-dependent mutant p53 activity for at least some REs and possibilities for reactivation of cancer mutants. We propose a general synergistic relationship between the p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. In collaboration with Alberto Inga (Trento University) and Ken Korach (NIEHS), we are evaluating effects of different ER mutants that affect DNA and accessory protein interactions on p53 mediated transactivation of genes with in cis ER and p53 REs. We developed a functional matrix tool for genome-wide searches for putative p53 target genes. Several new p53 target genes have been identified including the DNA repair-associated RAP80 gene as well as several members of the Toll-like receptors (TLRs) family that determine innate immunity (discussed below). As predicted for RAP80 as well as for several TLRs, including TLRs 2, 5 and 10, p53 and ER can cooperate to mediate its transcription both in cancer cell lines and human lymphocytes. CANCER-ASSOCIATED P53 MUTANTS. Nearly all cancers have mutant or reduced expression of the p53 tumor suppressor gene. Using yeast-based and human cell systems, we found that p53 mutations can lead to considerable diversity in the spectrum of responses from REs including 1) decrease/loss-of-function; 2) subtle changes; 3) altered specificity; and 4) super-transactivation all of which lead to variation in biological responses. Most of the functional mutants were able to function at 1/2 sites and several can cooperate with ERs to mediate expression of FLT1 revealing a new level of functional interaction between these two master regulators. With the inclusion of immune response-related TLR genes into the p53 network, we evaluated the effect of a panel of 25 tumor-associated p53 mutants on TLR gene family expression after transient transfection in p53 null cancer cell lines. Changes in TLR transactivation patterns, including change-of-spectrum were observed, suggesting that p53 tumor status might be an important factor in adjuvant therapy employing TLR pathways to treat cancer. P53 NETWORK EVOLUTION. We are investigating evolution of REs in terms of responsiveness to p53. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional REs during p53 network evolution. Only 1/3 of the REs found in humans are predicted to be functionally conserved in rodents. Importantly, we found functional conservation of weakly responding REs including 1/2 sites. Among validated p53 REs conserved between rodents and humans, one third were comprised of 1/2- or 3/4-sites, each with a perfect consensus -site suggesting a selective advantage in retaining weak p53 REs. Importantly, the integration of the TLR gene family into the p53 network also appears unique to primates. With our CHIP seq based whole genome p53 binding map generated in primary and cancer cell lines we are addressing evolutionary sequence conservation. REGULATION OF THE IMMUNE RESPONSE BY p53 The identification of p53 target REs associated with TLR genes has led to exciting new findings. Among the 10 human TLRs, nine had canonical and noncanonical p53 REs. Using primary human cells obtained in a collaboration with the Clinical Research Unit we examined expression of the entire TLR gene family following exposure to anti-cancer p53 inducing agents. Expressions of all TLR genes in blood lymphocytes and alveolar macrophages from healthy volunteers were inducible by DNA metabolic stressors. However, there is considerable inter-individual variability. Furthermore, a polymorphism in the TLR8 promoter provides the first human example of a p53 target RE sequence specifically responsible for endogenous gene induction. Similarly p53 dependent TLR expression is detected in human cancer cell lines. For some TLRs the p53 control seems to enhance the inflammatory responses, mediated by activation of TLRs in the presence of natural ligands. In particular, we found a p53-dependent increase in response to the TLR5 ligand flagellin as measured by both mRNA and protein production of the downstream cytokines IL-6 and IL-8 accompanied by a specific increase in phosphorylation of p38 MAP kinase which is one of the mediators of TLR signaling. We also found that in the absence of TLR ligands, induced p53 can cooperate with Nuclear Factor-kappa beta (NF-kB) to induce pro-inflammatory cytokines in primary human macrophages. DEVELOPMENT OF HIGH THROUGHPUT YEAST-BASED SYSTEM FOR p53 and NFKAPPAB. We are expanding our yeast based system to create high-throughput systems for addressing chemical modification of wild type and mutant human p53, factors that interact with p53 as well as adapting the system to address components of the NFkappa master regulator. The human proteins are expressed in yeast at varying levels and their ability to function as transcription factors at known target sequences is determined. Chemical modification is assessed as changes in expression of reporters.

p53肿瘤抑制剂和主调节剂是人类DNA修复，损害检查点和人类生物学许多方面的核心。重要的是，对于p53功能，大多数癌症都会改变。我们发现了p53目标和人类多样性的宇宙大大扩展，以及压力反应的变化。 p53的DNA结合和反式激活对于肿瘤抑制至关重要。 p53的靶向基因的p53依赖性表达有很大差异，从而导致p53介导的生物学后果差异。这应归因于目标响应元素（RE）序列的部分变化。 我们发现，目标序列基序与体外得出的RE共有目标序列有很大区别，该序列由2份RRRCA/TT/AGYYY组成，该副本由最多13个碱基的垫片分隔。我们专注于RE功能，即RES支持p53反式激活的能力。为了直接评估人类RES的反式激活反应性，我们开发了发芽的酵母中的启动子系统，用于可变的p53表达。这已被用来建立RES跨物种的功能演变，并在我们的钢琴模型中总结了描述转录网络中功能变异性的钢琴模型。我们已经将酵母中的许多人类p53转化为培养和离体中的人类细胞。 我们先前的研究表明，FLT1基因启动子中的SNP产生了完美的p53磁场，并启用了p53介导的FLT-1的反式激活，将VEGF系统直接放置在p53网络中。我们发现，通常在p53中可以通过包括位点在内的非规范结合序列进行反式激活。这一发现大大扩展了p53主监管网络。我们使用CHIP-seq分析扩展了这些研究，以鉴定p53结合位点和相关的基因表达变化，而在癌细胞中不同药物激活p53后，相关的基因表达变化。有趣的是，结合通常与反式激活无关。 对体内共识结合序列的从头序列搜索显示，p53规范基序实际上是2个串联rrrcwwgyyy decamers，没有间隔者。尽管效率较低，但p53可以结合由带有垫片或仅位点组成的非规范p53基序。 p53和ER调节网络的相互作用。我们已经确定了一个1/2个位点的雌激素受体RE，该RE大大增加了FLT1 1/2位点P53 RE的p53反式激活，从而确立了p53调节网络的新维度。最近，我们解决了p53和ER协同反式激活的一般性。在顺式中，配体激活的ER作用大大增强了p53的反式激活。增加的反式激活扩展到几个与癌症相关的p53突变体，这表明至少某些RES和癌症突变体重新激活的可能性依赖ER依赖性突变体p53活性。我们提出了p53家族与ER主调节剂之间的一般协同关系，在p53目标规范和非规范性RES的反式激活中可能对p53的响应不佳。与Alberto Inga（Trento University）和Ken Korach（NIEHS）合作，我们正在评估不同ER突变体的影响，这些突变体影响DNA和辅助蛋白相互作用对p53介导的基因的介导的基因的反式激活，该基因具有CIS ER和p53 Res。 我们开发了一个功能性矩阵工具，用于全基因组搜索推定的p53靶基因。已经确定了几种新的p53靶基因，包括DNA维修相关的RAP80基因以及确定先天免疫力的Toll样受体（TLR）家族的几个成员（下面讨论）。正如Rap80和几个TLR所预测的那样，包括TLRS 2、5和10，P53和ER可以合作以在癌细胞系和人淋巴细胞中介导其转录。 癌症相关的p53突变体。几乎所有癌症都具有p53肿瘤抑制基因的突变体或降低的表达。使用基于酵母的和人类细胞系统，我们发现p53突变可以导致RES的响应范围中的相当多样性，包括1）减少/功能丧失； 2）微妙的变化； 3）特异性改变； 4）超级转移所有这些都导致生物学反应的变化。大多数功能突变体能够在1/2个位点功能，并且一些可以与ERS合作介导FLT1的表达，从而揭示了这两个主调节剂之间的新功能相互作用。 随着与免疫反应相关的TLR基因纳入p53网络，我们评估了一组与p53无效细胞系短暂转染后25个肿瘤相关p53突变体对TLR基因家族表达的影响。观察到TLR反式激活模式的变化，包括光谱变化，表明p53肿瘤状态可能是使用TLR途径治疗癌症的辅助治疗的重要因素。 p53网络演变。我们正在研究RES的进化，以p53的响应能力。单个RES在p53网络演化过程中表现出明显的反式激活以及功能性RES的广泛周转率的明显差异。预计在人类中发现的RES中只有1/3在啮齿动物中具有功能保守。重要的是，我们发现功能保护对弱反应RES，包括1/2个地点。在啮齿动物和人类之间保守的经过验证的p53固定中，三分之一由1/2或3/4点组成，每个位置都有一个完美的共识 - 表明在保留弱p53 Res方面具有选择性优势。重要的是，将TLR基因家族的整合到p53网络中似乎也是灵长类动物的独特之处。通过在原始和癌细胞系中产生的基于芯片SEQ的整个基因组p53结合图，我们正在解决进化序列保护。 p53对免疫反应的调节 与TLR基因相关的p53目标RES的识别导致了令人兴奋的新发现。在10个人类TLR中，有9个具有规范和非规范p53 res。使用与临床研究单位合作获得的原代人细胞，我们检查了暴露于抗癌p53诱导剂后整个TLR基因家族的表达。 DNA代谢应激源可诱导血液淋巴细胞和肺泡巨噬细胞中所有TLR基因的表达。但是，存在相当大的个体差异。此外，TLR8启动子中的多态性提供了p53靶标的序列的第一个人类示例，这些序列是专门负责内源基因诱导的。 同样，在人类癌细胞系中检测到依赖性TLR表达。对于某些TLR，p53的控制似乎可以增强炎症反应，这是在天然配体存在下TLR的激活介导的。特别是，我们发现p53依赖性增加了对TLR5配体鞭毛蛋白的响应，通过mRNA和蛋白质产生下游细胞因子IL-6和IL-8的蛋白质产生，并伴随着p38 MAP激酶的磷酸化的特异性增加，这是TLR信号的中介体之一。我们还发现，在没有TLR配体的情况下，诱导的p53可以与核因子-kappa beta（NF-KB）合作，以诱导原代人巨噬细胞中促炎性细胞因子。 开发用于P53和NFKAPPAB的高吞吐量酵母系统。 我们正在扩展基于酵母的系统，以创建高通量系统，以解决野生型和突变体p53的化学修饰，与p53相互作用的因素以及调整系统以解决NFKAPPA主调节器的组件。 人蛋白在酵母中以不同的水平表达，并确定其作为转录因子在已知目标序列中发挥作用的能力。 化学修饰被评估为记者表达的变化。