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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

基本信息

批准号：
8777942
负责人：
Gregory M Gauthier
金额：
$ 18.81万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8777942
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 response transcription factor transmission process

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：在世界范围内，二型真菌每年造成数百万例感染。这些真菌在酵母（37 ℃）和霉菌（22 ℃）之间进行可逆转换。作为酵母的生长促进宿主免疫力的逃避以引起疾病，而作为霉菌的生长促进在土壤中的生存，通过有性繁殖的遗传多样性，以及传播到新的宿主。尽管热二型性的重要性，真菌如何调节温度适应的问题是知之甚少，代表了知识的一个主要空白。长期目标是描绘真菌适应温度的分子机制。该研究旨在研究Blastomeridermatification中的加塔转录因子SREB（Blastomeridermatification中的铁载体生物合成抑制因子）如何调控对温度的适应，以及这种调控是否与铁稳态有关。SREB无效突变体不能在22 ℃完成温度依赖性转化为霉菌，并且不能适当地调节铁稳态。虽然大多数研究都集中在温度从22摄氏度到37摄氏度的变化，但另一个方向的变化-37摄氏度到22摄氏度-却没有得到充分的重视。此外，下游靶基因和用于响应温度（37 ℃或22 ℃）的机制仍然不明确。使用基因表达微阵列分析SREB，发现该基因的缺失导致在37 ℃和22 ℃下转录的多效性变化。染色质免疫沉淀与定量实时PCR（ChIP-qPCR）表明，SREB结合基因的不同功能在37 ℃和22 ℃。此外，已经鉴定了SREB控制下的几个候选“非铁”和“铁”基因用于功能测试。假设SREB在加塔基序处结合DNA以调节基因转录，这反过来控制对温度的适应，这表现为向霉菌的转变。目的1：使用ChIP和DNA测序（ChIP-seq）在37 ℃和22 ℃下在全基因组范围内鉴定SREB结合的基因。ChIP-seq是高效的，并且允许识别SREB结合的基因，而不会偏向于特定的基序。当与基因表达微阵列和基序分析相结合时，ChIP-seq将提供关于SREB如何在37 ℃和22 ℃下影响转录的新的深入知识。目的2：功能测试我们“手头”的SREB结合基因（以及ChIP-seq鉴定的那些基因）对温度适应的影响（即，转化为模具）。候选基因“在手”将通过改变转录本丰度进行测试，并分析从37 ℃到22 ℃过渡期间的缺陷。通过ChIP-seq鉴定的其他SREB结合基因（“铁”和“非铁”）将被优先考虑并以类似的方式进行测试。这项研究是创新的，因为我们专注于一个未充分研究的，但二型性的组成部分，即向霉菌的过渡，以了解真菌如何适应温度。这项研究意义重大，因为结果将提供新的见解，并作为破译真菌适应温度的机制的基础。关于温度适应的基础研究具有长期潜力，可以为真菌感染患者阐明新的治疗策略。