Regulation of Morphogenesis in Dimorphic Fungi by a GATA Transcription Factor

描述（由申请人提供）：在世界范围内，二态真菌每年导致数百万人感染。这些真菌经历酵母（37℃）和霉菌（22℃）之间的可逆转变。作为酵母的生长促进逃避宿主的免疫从而引起疾病，而作为霉菌的生长促进土壤中的生存，通过有性繁殖促进遗传多样性，并传播给新的宿主。尽管热二态性的重要性，真菌如何调节温度适应的问题是知之甚少，代表了一个主要的知识缺口。长期目标是描述真菌适应温度的分子机制。本研究拟探讨皮炎芽孢菌中的GATA转录因子SREB （Blastomyces siderophore biosynthesis repressor in Blastomyces）如何调控温度适应，以及这种调控是否与铁稳态有关。SREB零突变体在22℃时不能完成温度依赖的向霉菌的转化，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摄氏度的变化上，但另一个方向的变化——从37摄氏度到22摄氏度——却被低估了。此外，用于响应温度（37℃或22℃）的下游靶基因和机制仍然不明确。基因表达芯片分析显示，在37℃和22℃时，SREB基因缺失导致转录发生多效性变化。染色质免疫沉淀定量实时PCR （ChIP-qPCR）显示SREB在37℃和22℃下结合不同功能的基因。此外，在SREB控制下的几个候选“非铁”和“铁”基因已被确定用于功能测试。假设SREB结合GATA基序上的DNA来调节基因转录，这反过来又控制了对温度的适应，这种适应表现为向霉菌的转变。目的1：利用ChIP和DNA测序（ChIP-seq）在37℃和22℃的全基因组范围内鉴定SREB在体内结合的基因。ChIP-seq是高效的，可以识别sreb结合的基因，而不偏向于特定的基序。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在37℃和22℃下如何影响转录的新的深入知识。目标2：对sreb结合基因（以及通过ChIP-seq鉴定的基因）进行功能测试，以了解它们对温度适应（即转化为霉菌）的影响。候选基因“在手”将通过改变转录物丰度来测试，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”）将优先排序并以类似的方式进行测试。这项研究是创新的，因为我们关注的是一个未被充分研究的，但却是二态性的一个组成部分，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，并为破译真菌适应温度的机制奠定基础。温度适应的基础研究具有长期潜力，可以为真菌感染患者提供新的治疗策略。

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.