Aflatoxin Regulation: Role of Global Regulators and Epigenetic Phenomena

说明(申请人提供)：黄曲霉毒素(AFS)是由人类病原体黄曲霉和寄生曲霉产生的致癌和致畸作用最强的次生代谢物之一。这些无处不在的真菌感染我们的食品和农作物，并产生AFs，一旦摄入，就会形成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灭产AF的真菌，因此迫切需要解决AF污染问题。这项研究的主要目的是扩大我们在房颤产生的遗传调控领域的知识。最近，在一种以杂色曲霉毒素(ST，房颤的晚期前体)为SM的相关真菌Nidulans中发现了一种核蛋白和全球次生代谢调节因子LaeA。在正在进行的研究中，我们从产生AF的黄曲霉中分离出了laeA同源基因。LeeA的缺失会导致AFS的丧失、感染性的降低和菌核(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房颤阴性、发育改变的寄生虫SEC-(次生代谢减去)变体重叠。 在这个方案中，我们将检验三个假设：(1)房颤阴性的sec表型涉及laeA表达的变化。为了验证这一假设，我们将进行实验，在SEC-菌株中过度表达和删除leeA。(2)LaeA通过与房颤途径特异性调节因子和/或其他染色质蛋白相互作用来影响房颤的产生。为了验证这一假设，我们将使用酵母双杂交试验。(3)次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，更有可能面临染色体损伤的风险，特别是在亚端粒区域，这是一个包含AF簇的区域，容易受到遗传异常的影响。为了测试表观遗传学的变化，我们将检查DNA甲基化和转座子位置在秒突变中的变化。这些研究的结果将更好地确定全球转录调节因子(LAEA)在房颤产生和次生代谢中的作用。从长远来看，这可能有助于研究人员开发更好的策略(例如，鉴定天然抑制剂和开发基因工程菌株)来减少房颤污染。这项研究的知识还可能对以发育异常、染色体异常和表观遗传学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.