Functional divergence of a global regulatory complex governing fungal filamentation. Functional divergence of a global regulatory complex governing fungal filamentation

Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to enable transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Epistasis experiments suggested that Mfg1 acts downstream of protein kinase A, as does Flo8. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Mfg1 and Flo8 in C. albicans. A key direct transcriptional target of Mfg1 is TEC1, for which overexpression was sufficient to restore filamentation in the absence of Mfg1. To identify additional Mfg1 downstream effectors, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the amplification on chromosome 6 is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis. Overall design: We performed chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) with strains harboring Flo8 or Mfg1 epitope-tagged with a tandem affinity purification (TAP) epitope in yeast (YPD 30°C) and hyphal condition (YPD 37°C). To identify the Flo8 and Mfg1 targets that are also transcriptionally modulated by these regulators, we performed microarray analysis comparing the gene expression profiles of mfg1Δ/mfg1Δ, flo8Δ/flo8Δ and flo8Δ/flo8Δ mfg1Δ/mfg1Δ mutants with a wild-type strain under basal (yeast condition) and filament-inducing conditions

形态发生转变在真菌界中普遍存在。对于重要的人类致病真菌白色念珠菌（Candida albicans）而言，在酵母态与菌丝态之间转换的能力是其致病力的关键。而模式酿酒酵母（Saccharomyces cerevisiae）的菌丝形成能力则有助于其获取营养物质。近期一项针对酿酒酵母的功能基因组筛选研究，将Mfg1鉴定为形态发生的调控因子，它可与Flo8及Mss11形成复合物，介导对菌丝形成至关重要的转录应答。在白色念珠菌中，Mfg1同样可与Flo8和Mss11发生物理相互作用，且对于响应多种信号的菌丝形成过程不可或缺，但其调控形态发生的具体分子机制仍未明确。
本研究探究了扰动Mfg1、Flo8及Mss11对白色念珠菌形态发生的影响，并鉴定出该复合物各成员存在功能分化。我们发现，白色念珠菌的Mss11对于菌丝形成并非必需，且即便在Mfg1缺失的情况下，FLO8的过表达也可诱导持续性的菌丝形成。上位性实验结果表明，Mfg1与Flo8一样，均作用于蛋白激酶A的下游。通过结合转录谱分析与染色质免疫共沉淀芯片（ChIP-chip）技术，我们鉴定出白色念珠菌中Mfg1与Flo8的转录靶标存在差异。Mfg1的关键直接转录靶标为TEC1，过表达该基因即可在Mfg1缺失的情况下恢复菌丝形成能力。为了鉴定更多Mfg1下游的效应因子，我们采用了一种全新的策略，筛选能够在Mfg1缺失时恢复菌丝形成的突变体。对具备菌丝形成能力的突变体进行全基因组测序后发现，6号染色体扩增是一种保守的适应性机制。该6号染色体扩增的关键决定因素为FLO8：若缺失其中一个等位基因则会阻断形态发生，且在将FLO8重新定位至其他染色体的进化谱系中，并未出现6号染色体的扩增。因此，本研究揭示了关键形态发生调控因子在进化过程中的重布线现象，以及非整倍体作为驱动真菌形态发生的适应性机制。
整体实验设计：我们分别在酵母态培养条件（YPD培养基、30℃）与菌丝诱导条件（YPD培养基、37℃）下，对带有串联亲和纯化（TAP）标签的Flo8或Mfg1菌株开展染色质免疫共沉淀芯片（ChIP-chip）实验。为了鉴定同时受这些调控因子转录调控的Flo8与Mfg1靶标基因，我们在基础培养条件（酵母态）与菌丝诱导条件下，分别将mfg1Δ/mfg1Δ、flo8Δ/flo8Δ以及flo8Δ/flo8Δ mfg1Δ/mfg1Δ突变体的基因表达谱与野生型菌株进行对比分析。