Chemical stimuli override a temperature-dependent morphological program by reprogramming the transcriptome

The human fungal pathogen Histoplasma changes its morphology in response to temperature. At 37°C it grows as a budding yeast whereas at room temperature it transitions to hyphal growth. Prior work has demonstrated that 15-20% of transcripts are temperature-regulated, and that transcription factors Ryp1-4 are necessary to establish yeast growth. However, little is known about transcriptional regulators of the hyphal program. To identify TFs that regulate filamentation, we utilize chemical inducers of hyphal growth. We show that addition of cAMP analogs or an inhibitor of cAMP breakdown overrides yeast morphology, yielding inappropriate hyphal growth at 37°C. Additionally, butyrate supplementation triggers hyphal growth at 37°C. Transcriptional profiling of cultures filamenting in response to cAMP or butyrate reveals that a limited set of genes respond to cAMP while butyrate dysregulates a larger set. Comparison of these profiles to previous temperature- or morphology-regulated gene sets identifies a small set of morphology-specific transcripts. This set contains 9 TFs of which we characterized three, STU1, FBC1, and PAC2, whose orthologs regulate development in other fungi. We found that each of these TFs is individually dispensable for room-temperature (RT) induced filamentation but each is required for other aspects of RT development. FBC1 and PAC2, but not STU1, are necessary for filamentation in response to cAMP . Ectopic expression of each of these TFs is sufficient to induce filamentation at 37°C. Finally, PAC2 induction of filamentation at 37°C is dependent on STU1, suggesting these TFs form a regulatory circuit that, when activated at RT, promotes the hyphal program. Expression profiling by RNA-seq of Histoplasma yeast cells treated with cAMP analogs or butyrate with three bioreps per treatment.

人类致病真菌荚膜组织胞浆菌（Histoplasma）可响应温度变化改变自身形态：在37℃条件下以出芽酵母态生长，而在室温环境中则转换为菌丝生长模式。既往研究已证实，15%~20%的转录本受温度调控，且转录因子Ryp1-4是建立酵母态生长的必需因子。然而，目前对菌丝生长程序的转录调控因子仍所知甚少。
为鉴定调控菌丝形成的转录因子，本研究采用了菌丝生长的化学诱导剂。实验结果显示，添加cAMP类似物或cAMP分解抑制剂可逆转酵母态形态，使37℃下出现异常的菌丝生长；此外，补充丁酸盐同样可在37℃下触发菌丝生长。对经cAMP或丁酸盐诱导形成菌丝的培养物进行转录组分析后发现，仅有限的基因集合响应cAMP调控，而丁酸盐则可调控更大规模的基因集。将上述转录谱与既往温度或形态调控基因集进行比对，可鉴定出一小类形态特异性转录本。该基因集包含9个转录因子，我们对其中3个——STU1、FBC1和PAC2——开展了功能验证，这三个转录因子的同源基因在其他真菌中可调控发育过程。研究发现，这三个转录因子各自对于室温诱导的菌丝形成均非必需，但均参与了室温下发育的其他过程。FBC1与PAC2（而非STU1）是cAMP诱导菌丝形成的必需因子；在37℃下异位表达这三个转录因子中的任意一个，均足以诱导菌丝形成。最后，PAC2在37℃下诱导菌丝形成的过程依赖于STU1，这表明这些转录因子构成了一个调控回路，当在室温下被激活时，该回路可促进菌丝生长程序。
本研究通过RNA-seq对经cAMP类似物或丁酸盐处理的荚膜组织胞浆菌酵母细胞进行了表达谱分析，每个处理设置三个生物学重复。