Genetic analysis of pleiotropic drug resistance

念珠菌是第四种最常见的血液感染类型，仅有三种 常用的抗真菌药物可用，治疗选择受到耐药性的威胁。 与光滑念珠菌有关的感染是第二种最常见的念珠菌血症类型， 自本世纪初以来一直在增加。光滑线虫对唑类具有较强的抗药性。 药物，最常用的一类抗真菌化合物。对唑类有抗药性的菌株有 通常被发现在PDR1基因中包含功能增益(GOF)突变，编码一种转录 这一因素是耐药性的核心调节因素。Pdr1的这些GOF形式使其结构性升高 靶基因的转录水平。其中最核心的是三磷酸腺苷结合盒运输器- 编码CDR1基因座。Cdr1是一种药物外排泵，可防止体内有毒的唑类药物积聚。 牢房。唑类药物的靶标是由ERG11编码的羊毛甾醇-14去甲基酶 吉恩。唑类药物激发通过Upc2a的作用诱导ERG11转录 转录因子。由Pdr1和Upc2A定义的转录调控电路之前 被视为对唑类耐药的单独途径。我们最近发现Upc2A控制着 PDR1和CDR1的转录，表明它们之间存在生理联系 监管网络。在这项提案中，我们计划剖析Pdr1激活Pdr1的机制(S 基因表达，并在靶基因DNA水平上剖析Pdr1与Upc2a的相互作用 装订和转录。在目标1中，我们将确定辅助激活剂的功能贡献 我们通过质谱学发现的与Pdr1共纯化的蛋白质。表位标记与基因 干扰等位基因将允许确定这些蛋白质如何相互作用并调节 Pdr1调节基因表达。AIM 2将识别与Pdr1 C末端相互作用的蛋白质靶点 负责招募转录介体的转录激活结构域 基因表达的复杂性和诱导性。我们还将使用交联法来鉴定蛋白质。 与这个结构域以及Pdr1中潜在的负调控结构域相互作用。最后，我们 将使用染色质免疫沉淀与高通量测序和 RNA测序以确定Pdr1和Upc2A对共同调控基因的相互影响。我们会 也干扰了受这两个因子调控的其他转录因子基因，以确定更大的 参与唑类药物挑战转录反应的基因。我们发现的联系 Pdr1和Upc2a之间的关系表明，这些因素协同作用，使唑类耐药性成为 它们的正常功能。对这种合作的分子基础的理解将提供对 可用于减弱抗药性的新漏洞。

Candidemias represent the 4th most common type of bloodstream infection and with only three commonly used antifungal drugs available, treatment options are threatened by drug resistance. Infections linked to Candida glabrata represent the second most common type of candidemia and have been increasing since the early 2000s. C. glabrata exhibits a robust ability to acquire resistance to azole drugs, the most commonly used class of antifungal compounds. Isolates that are azole resistant are routinely found to contain gain-of-function (GOF) mutations in the PDR1 gene, encoding a transcription factor that is a central regulator of drug resistance. These GOF forms of Pdr1 drive constitutively high levels of transcription of target genes. Central among these is the ATP-binding cassette transporter- encoding CDR1 locus. Cdr1 is a drug efflux pump that prevents the accumulation of toxic azole levels in the cell. The target of azole drugs is the lanosterol -14 demethylase enzyme encoded by the ERG11 gene. ERG11 transcription is induced by azole drug challenge through the action of the Upc2A transcription factor. The transcriptional regulatory circuits defined by Pdr1 and Upc2A have previously been treated as separate pathways to azole resistance. We recently discovered that Upc2A controls transcription of both PDR1 and CDR1, indicating the presence of a physiological tie between these regulatory networks. In this proposal, we plan on dissecting the mechanism(s) used by Pdr1 to activate gene expression and dissect the interaction between Pdr1 and Upc2A at the levels of target gene DNA- binding and transcription. In aim 1, we will determine the functional contribution made by coactivator proteins that we have found by mass spectrometry to co-purify with Pdr1. Epitope-tagging and gene disruption alleles will allow determination of how these proteins interact with and modulate the ability of Pdr1 to regulate gene expression. Aim 2 will identify protein targets interacting with the Pdr1 C-terminal transcriptional activation domain that are responsible for recruitment of the transcriptional Mediator complex and induction of gene expression. We will also use cross-linking approaches to identify proteins that interact with this domain as well as potential negative regulatory domains within Pdr1. Finally, we will use a combination of chromatin immunoprecipitation coupled with high-throughput sequencing and RNA-sequencing to determine the mutual impact Pdr1 and Upc2A have on co-regulated genes. We will also disrupt other transcription factor genes regulated by these two factors to identify the larger suite of genes involved in the transcriptional response to azole drug challenge. Our finding of the connections between Pdr1 and Upc2A indicates that these factors cooperate to confer azole resistance as part of their normal function. Understanding of the molecular basis of this cooperation will provide insight into new vulnerabilities to use to attenuate drug resistance.