Regulation of Morphogenesis in Dimorphic Fungi by a GATA Transcription Factor

摘要：在世界范围内，双相真菌每年造成数百万人感染。这些真菌经历了 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 免疫力导致疾病，而霉菌的生长促进了土壤中的生存，遗传多样性通过性 繁殖和传播到新的宿主。尽管热二态性的重要性， 真菌如何调节温度适应性知之甚少，代表了知识上的一个重大空白。的 长期目标是描述真菌适应温度的分子机制。研究 提出研究如何加塔转录因子在芽生菌皮炎，SREB（铁载体 生物合成阻遏物），支配对温度的适应，以及这种调节是否是 与铁平衡有关SREB无效突变体不能完成温度依赖性转化， 霉菌在22 oC和不能适当地调节铁稳态。虽然大多数研究都集中在 当温度从22摄氏度变化到37摄氏度时，另一个方向的变化--37摄氏度到22摄氏度--被低估了。 此外，下游靶基因和机制用于响应温度（37 ℃或22 ℃） 仍然不明确。使用基因表达微阵列分析SREB显示该基因的缺失 在37 ℃和22 ℃引起转录的多效性变化。染色质免疫沉淀定量 实时PCR（ChIP-qPCR）证实SREB在37 ℃和22 ℃下结合具有不同功能的基因。 此外，已经鉴定了SREB控制下的几个候选“非铁”和“铁”基因， 功能测试假设SREB在加塔基序处结合DNA以调节基因转录， 转，控制着对温度的适应，这表现在向模具的转变。目标1：识别基因 使用ChIP和DNA测序（ChIP-seq），SREB在37 ℃和22 ℃下以全基因组规模在体内结合。 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将提供新的，深入的 了解SREB在37 ℃和22 ℃下如何影响转录。目标2：SREB结合的功能测试 我们已经“掌握”的基因（以及ChIP-seq鉴定的那些）对温度适应的影响（即， 转化为模具）。“手头”的候选基因将通过改变转录本丰度来测试并分析 在从37 oC到22 oC的转变过程中，其他SREB结合基因（“铁”和“非铁”） ChIP-seq鉴定的基因将被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 正专注于一个未被充分研究，但不可分割的一部分，二型性，过渡到模具，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的见解， 作为破解真菌适应温度机制的基础。温度基础研究 适应具有阐明真菌感染患者的新治疗策略的长期潜力。

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.