Aflatoxin Regulation: Role of Global Regulators and Epigenetic Phenomena

描述（由申请方提供）：黄曲霉毒素（AF）是由人类病原体黄曲霉和A.寄生虫这些无处不在的真菌感染我们的食品和作物，并产生AF，在摄入后，形成AF/DNA加合物，损害免疫力并导致肝癌。由于不可能从自然界中消除产生AF的真菌，因此非常需要解决AF污染的问题。本研究的主要目的是扩大我们的知识在该领域的遗传调控AF生产。LaeA是一种核蛋白，是植物次生代谢的全局调节因子。nidulans是一种以杂色曲霉素（ST，AF的晚期前体）为SM的相关真菌。在正在进行的研究中，我们已经在产生AF的A.黄的laeA的缺失导致AF的丧失、感染性降低和菌核（与AF产生相关的发育结构）的丧失。这些特征与我们实验诱导的AF阴性、发育改变的A。寄生虫sec-（次级代谢减）变体。 在这个建议中，我们将测试三个假设：（1）AF阴性sec-表型涉及laeA表达的变化。为了检验这一假设，我们将进行实验以在sec-菌株中过表达和缺失laeA。(2)LaeA通过与AF通路特异性调节剂和/或与其他染色质蛋白相互作用来对AF产生发挥作用。为了验证这一假设，我们将使用酵母双杂交试验。(3)sec-表型是由表观遗传事件引起的。sec-菌株绕过了某些发育事件，更有可能处于染色体损伤的风险中，特别是在亚端粒区域，该区域包含AF簇并且对遗传异常敏感。为了测试表观遗传变化，我们将检测sec突变体中DNA甲基化和转座子位置的变化。这些研究的结果将更好地定义全局转录调节因子（LaeA）对AF产生和次级代谢的作用。从长远来看，这可能有助于研究人员制定更好的策略（例如，天然抑制剂的鉴定和基因工程菌株的开发）以减少AF污染。这项研究的知识也可能对以异常发育、染色体畸变和表观遗传变化为特征的两个领域产生更广泛的影响;即衰老和癌症。

DESCRIPTION (provided by applicant): Aflatoxins (AFs) are among the most carcinogenic and teratogenic secondary metabolites (SMs) produced by the human pathogen Aspergillus flavus and A. parasiticus. These ubiquitous fungi infect our food products and crops and produce AFs which, upon ingestion, form AF/DNA adducts, impair immunity and cause liver cancer. Since it is impossible to eliminate the AF-producing fungi from nature, there is a great need to address the problem of AF contamination. The major goal of this study is to expand our knowledge n the field of genetic regulation of AF production. Recently, LaeA, a nuclear protein and a global regulator of secondary metabolism, was discovered in A. nidulans, a related fungus making sterigmatocystin (ST, a late precursor of AF) as its SM. In ongoing studies, we have isolated the laeA ortholog in the AF-producing A. flavus. Deletion of laeA results in loss of AFs, reduced infectivity and loss of sclerotia (developmental structures associated with AF production). These characteristics overlap with our experimentally induced AF-negative, developmentally altered A. parasiticus sec- (for secondary metabolism minus) variants. In this proposal we will test three hypotheses: (1) The AF-negative sec- phenotype involves changes in laeA expression. To test this hypothesis, we will carry out experiments to both over-express and delete laeA in the sec- strains. (2) LaeA exerts its effects on AF production by interacting with AF pathway-specific regulators and/or with other chromatin proteins. To test this hypothesis, we will use yeast two-hybrid assays. (3) The sec- phenotype is caused by epigenetic events. The sec- strains, which have bypassed certain developmental events, are more likely at risk for chromosomal damage, especially in the sub-telomeric region, a region which harbors the AF cluster and is susceptible to genetic abnormalities. To test for epigenetic changes, we will examine changes in DNA methylation and transposon position in sec- mutants. Results from these studies will better define the role of global transcriptional regulators (LaeA) on AF production and secondary metabolism in general. Long-term, this may assist researchers to develop better strategies (e.g., identification of natural inhibitors and development of genetically engineered strains) to reduce AF contamination. Knowledge from this study may also have a broader impact on two areas characterized by abnormal development, chromosomal aberrations and epigenetic changes; namely, aging and cancer.