Regulation of Morphogenesis in Dimorphic Fungi by a GATA Transcription Factor

GATA 转录因子对二态真菌形态发生的调节

• 批准号: 8636380
• 负责人: Gregory M Gauthier
• 金额: $ 22.58万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636380
• 关键词: Affect; Anabolism; Animal Model; Basic Science; Binding; Binding Sites; Biology; Blastomyces; Blastomyces dermatitidis; Candidate Disease Gene; Categories; Cryptococcus neoformans; DNA; DNA Binding; DNA Sequence; DNA Sequence Analysis; Data; Defect; Development; Disease; Event; Foundations; Gene Chips; Gene Deletion; Gene Expression Microarray Analysis; Gene Targeting; Generations; Genes; Genetic Transcription; Genetic Variation; Goals; Growth; Hand; Health; Histoplasma capsulatum; Homeostasis; Homologous Gene; Hour; Human; Immune system; Immunity; Infection; Iron; Knowledge; Life Style; Link; Molds; Molecular; Morphogenesis; Morphology; Mycoses; Ontology; Oxidation-Reduction; Partner in relationship; Pathogenesis; Patients; Persons; Phase Transition; Pneumonia; Process; Regulation; Reporter; Reproduction; Research; Saccharomycetales; Siderophores; Soil; Staging; Temperature; Testing; Therapeutic; Time; Transcript; Virulent; Yeasts; chromatin immunoprecipitation; dimorphism; fatty acid metabolism; fungus; genome-wide; human tissue; in vivo; innovation; insight; mutant; novel; novel therapeutics; overexpression; pathogen; public health relevance; response; transcription factor; transmission process

ABSTRACT: Worldwide, the dimorphic fungi cause several million infections each year. These fungi undergo a reversible transition between yeast (37oC) and mold (22oC). Growth as yeast promotes evasion of host immunity to cause disease, whereas growth as mold promotes survival in soil, genetic diversity through sexual reproduction, and transmission to new hosts. Despite the importance of thermal dimorphism, the question of how fungi regulate temperature adaptation is poorly understood and represents a major gap in knowledge. The long-term goal is to delineate the molecular mechanism(s) used by fungi to adapt to temperature. The research proposed investigates how a GATA transcription factor in Blastomyces dermatitidis, SREB (siderophore biosynthesis repressor in Blastomyces), governs the adaptation to temperature, and whether this regulation is linked with iron homeostasis. SREB null mutants fail to complete the temperature-dependent conversion to mold at 22oC and cannot properly regulate iron homeostasis. While most research has focused on the temperature change from 22oC to 37oC, the shift in the other direction - 37oC to 22oC - is underappreciated. Moreover, the downstream target genes and mechanisms used to respond to temperature (37oC or 22oC) remain ill defined. Analysis of SREB using gene expression microarrays revealed that deletion of this gene caused pleiotropic changes in transcription at 37oC and 22oC. Chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR) demonstrated SREB binds genes with disparate functions at 37oC and 22oC. Moreover, several candidate "non-iron" and "iron" genes under the control of SREB have been identified for functional testing. The hypothesis is SREB binds DNA at GATA motifs to regulate gene transcription, which in turn, controls the adaptation to temperature that is manifested by the transition to mold. Aim 1: Identify genes SREB binds in vivo on a genome-wide scale at 37oC and 22oC using ChIP with DNA sequencing (ChIP-seq). ChIP-seq is highly efficient and allows identification of SREB-bound genes without bias to specific motifs. When integrated with gene expression microarray and motif analyses, ChIP-seq will provide new, in-depth knowledge about how SREB impacts transcription at 37oC and 22oC. Aim 2: Functionally test SREB-bound genes we have "in-hand" (and those identified by ChIP-seq) for their impact on temperature adaptation (i.e., conversion to mold). Candidate genes "in-hand" will be tested by altering transcript abundance and analyzed for defects during the transition from 37oC to 22oC. Additional SREB-bound genes ("iron" and "non-iron") identified by ChIP-seq will be prioritized and tested in a similar fashion. The research is innovative because we are focusing on an understudied, but integral part of dimorphism, the transition to mold to understand how fungi adapt to temperature. The research is significant because the results will provide novel insight and serve as a foundation to decipher mechanisms used by fungi to adapt to temperature. Basic research on temperature adaptation has long-term potential to illuminate new therapeutic strategies for patients with fungal infections.

摘要：在世界范围内，二形真菌每年造成数百万次感染。这些真菌经历了 酵母 (37oC) 和霉菌 (22oC) 之间的可逆转变。酵母的生长促进宿主的逃避 免疫力导致疾病，而霉菌的生长促进土壤中的生存，通过性传播遗传多样性 繁殖，并传播到新的宿主。尽管热二态性很重要，但问题 人们对真菌如何调节温度适应知之甚少，这是一个重大的知识空白。这 长期目标是描绘真菌适应温度的分子机制。研究 提议研究皮炎芽生菌中的 GATA 转录因子 SREB（铁载体 芽生菌中的生物合成阻遏蛋白），控制对温度的适应，以及这种调节是否 与铁稳态有关。 SREB 无效突变体无法完成温度依赖性转化 在 22oC 下发霉，无法正确调节铁稳态。虽然大多数研究都集中在 温度从 22oC 变化到 37oC，但另一个方向的变化——37oC 到 22oC——却被低估了。 此外，用于响应温度（37oC或22oC）的下游靶基因和机制 仍然不明确。使用基因表达微阵列对 SREB 进行分析表明，该基因的缺失 在 37°C 和 22°C 下引起转录的多效性变化。染色质免疫沉淀定量 实时 PCR (ChIP-qPCR) 证明 SREB 在 37oC 和 22oC 下结合具有不同功能的基因。 此外，在 SREB 控制下的几个候选“非铁”和“铁”基因已被鉴定为 功能测试。假设 SREB 在 GATA 基序处结合 DNA 来调节基因转录，这在 转，控制对温度的适应，这通过向模具的过渡而体现出来​​。目标 1：识别基因 使用 ChIP 和 DNA 测序 (ChIP-seq)，SREB 在 37oC 和 22oC 下在体内在全基因组范围内结合。 ChIP-seq 非常高效，并且可以在不偏向特定基序的情况下识别 SREB 结合基因。 当与基因表达微阵列和基序分析集成时，ChIP-seq 将提供新的、深入的分析 关于 SREB 如何在 37oC 和 22oC 下影响转录的知识。目标 2：功能测试 SREB 绑定 我们“掌握”的基因（以及通过 ChIP-seq 鉴定的基因）对温度适应的影响（即， 转换为模具）。将通过改变转录物丰度来测试“现有”候选基因并进行分析 针对从 37oC 过渡到 22oC 期间的缺陷。其他 SREB 结合基因（“铁”和“非铁”） ChIP-seq 鉴定的结果将以类似的方式进行优先级排序和测试。这项研究具有创新性，因为我们 正在关注二态性的一个未被充分研究但不可或缺的部分，即向模具的过渡，以了解如何 真菌适应温度。这项研究意义重大，因为其结果将提供新颖的见解并服务于 作为破译真菌适应温度机制的基础。温度基础研究 适应具有为真菌感染患者阐明新治疗策略的长期潜力。