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 肿瘤抑制因子和主调节因子对于人类 DNA 修复、损伤检查点和人类生物学的许多方面至关重要。重要的是，大多数癌症的 p53 功能都发生了改变。我们发现了 p53 靶标范围的极大扩展、人类多样性以及应激反应的变化。 p53 的 DNA 结合和反式激活对于肿瘤抑制至关重要。数百个靶基因的 p53 依赖性表达存在相当大的差异，导致 p53 介导的生物学后果存在差异。这部分是由于目标响应元件 (RE) 序列的变化造成的。 我们发现靶序列基序与体外衍生的 RE 共有靶序列有很大不同，该序列由 2 个 RRRCA/TT/AGYYY 拷贝组成，由最多 13 个碱基的间隔区隔开。我们重点关注 RE 功能，即 RE 支持 p53 反式激活的能力。为了直接评估人类 RE 的反式激活反应性，我们在芽殖酵母中开发了用于可变 p53 表达的启动子系统。这已被用来建立跨物种 RE 的功能进化，并在我们的钢琴模型中进行了总结，该模型描述了转录网络内的功能变异性。我们已将酵母中人类 p53 的许多发现转化为培养和离体的人类细胞。 我们之前的研究表明，Flt1 基因启动子中的 SNP 会生成完美的 p53 位点，并使 p53 介导的 FLT-1 反式激活成为可能，从而将 VEGF 系统直接置于 p53 网络中。我们发现，一般来说，p53 可以通过非规范结合序列（包括位点）提供反式激活。这一发现极大地扩展了 p53 主调控网络。我们使用 ChIP-Seq 分析将这些研究扩展到全基因组范围，以确定癌细胞中 p53 被不同药物激活后的 p53 结合位点和相关基因表达变化。有趣的是，结合通常与反式激活无关。 对体内共有结合序列的从头基序搜索表明，p53 规范基序实际上是 2 个串联的 RRRCWWGYYY 十聚体，没有间隔区。尽管效率较低，p53 可以结合由带有间隔区的两个十聚体或仅一个位点组成的非规范 p53 基序。 p53 和 ER 监管网络的相互作用。我们发现了一种 1/2 位点雌激素受体 RE，它极大地增加了 FLT1 1/2 位点 p53 RE 上的 p53 反式激活，从而为 p53 调控网络建立了一个新的维度。最近，我们讨论了 p53 和 ER 协同反式激活的一般性。配体激活的 ER 以顺式作用大大增强了 p53 反式激活。反式激活的增加延伸到了几种与癌症相关的 p53 突变体，表明至少某些 RE 具有 ER 依赖性突变体 p53 活性，并且有可能重新激活癌症突变体。我们提出了 p53 家族和 ER 主调节因子在 p53 靶标典型和非典型 RE 反式激活中的一般协同关系，这些 RE 本身可能对 p53 反应较差。我们与 Alberto Inga（特伦托大学）和 Ken Korach（NIEHS）合作，正在评估不同 ER 突变体对 DNA 和辅助蛋白相互作用对 p53 介导的顺式 ER 和 p53 RE 基因反式激活的影响。 我们开发了一种功能矩阵工具，用于在全基因组范围内搜索假定的 p53 靶基因。几个新的 p53 靶基因已被鉴定，包括 DNA 修复相关的 RAP80 基因以及决定先天免疫的 Toll 样受体 (TLR) 家族的几个成员（如下所述）。正如对 RAP80 以及几种 TLR（包括 TLR 2、5 和 10）的预测，p53 和 ER 可以合作介导其在癌细胞系和人类淋巴细胞中的转录。 癌症相关的 P53 突变体。几乎所有癌症都有 p53 肿瘤抑制基因的突变或表达减少。使用基于酵母和人类的细胞系统，我们发现 p53 突变可以导致 RE 的反应谱出现相当大的多样性，包括 1）功能减少/丧失； 2）细微的变化； 3）改变特异性； 4）超级反式激活，所有这些都会导致生物反应的变化。大多数功能突变体能够在 1/2 位点发挥作用，并且一些可以与 ER 合作介导 FLT1 的表达，揭示了这两个主调节因子之间新水平的功能相互作用。 通过将免疫反应相关的 TLR 基因纳入 p53 网络，我们评估了一组 25 个肿瘤相关 p53 突变体在 p53 无效癌细胞系中瞬时转染后对 TLR 基因家族表达的影响。观察到 TLR 反式激活模式的变化，包括光谱的变化，表明 p53 肿瘤状态可能是利用 TLR 途径治疗癌症的辅助治疗的一个重要因素。 P53 网络演进。我们正在研究 RE 在对 p53 的响应方面的进化。在 p53 网络进化过程中，各个 RE 在潜在反式激活以及功能性 RE 的广泛更新方面表现出显着差异。据预测，在人类中发现的 RE 中，只有 1/3 在啮齿类动物中具有功能保守性。重要的是，我们发现弱响应 RE 的功能保守，包括 1/2 位点。在啮齿动物和人类之间保守的经过验证的 p53 RE 中，三分之一由 1/2 或 3/4 位点组成，每个位点都有一个完美的共有位点，表明在保留弱 p53 RE 方面具有选择性优势。重要的是，TLR 基因家族整合到 p53 网络中似乎也是灵长类动物所独有的。通过我们在原代细胞系和癌细胞系中生成的基于 CHIP seq 的全基因组 p53 结合图谱，我们正在解决进化序列保守问题。 p53 对免疫反应的调节 与 TLR 基因相关的 p53 靶 RE 的鉴定带来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 RE。使用与临床研究单位合作获得的原代人类细胞，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。健康志愿者的血液淋巴细胞和肺泡巨噬细胞中所有 TLR 基因的表达均可被 DNA 代谢应激源诱导。然而，个体之间存在相当大的差异。此外，TLR8启动子中的多态性提供了第一个专门负责内源基因诱导的p53靶RE序列的人类例子。 类似地，在人类癌细胞系中检测到 p53 依赖性 TLR 表达。对于某些 TLR，p53 控制似乎增强了炎症反应，这是通过在天然配体存在的情况下激活 TLR 介导的。特别是，通过下游细胞因子 IL-6 和 IL-8 的 mRNA 和蛋白质产量测量，我们发现对 TLR5 配体鞭毛蛋白的反应出现 p53 依赖性增加，同时伴随着 p38 MAP 激酶磷酸化的特异性增加，p38 MAP 激酶是 TLR 信号传导的介质之一。我们还发现，在缺乏TLR配体的情况下，诱导的p53可以与核因子-κβ（NF-kB）协同诱导原代人巨噬细胞中的促炎细胞因子。 针对 p53 和 NFKAPPAB 的高通量酵母系统的开发。 我们正在扩展基于酵母的系统，以创建高通量系统，用于解决野生型和突变型人类 p53 的化学修饰、与 p53 相互作用的因子，以及调整系统以解决 NFkappa 主调节因子的组件问题。 人类蛋白质在酵母中以不同的水平表达，并且确定了它们在已知靶序列上作为转录因子发挥作用的能力。 化学修饰被评估为报告基因表达的变化。