Transcriptional memory drives accelerated re-activation of secondary metabolite production in Aspergillus nidulans [ChIP-seq]

Organisms are repeatedly exposed to fluctuating environmental and nutritional conditions. Transcriptional memory has been shown to be a mechanism to cope with these fluctuations because it increases the speed and the magnitude of the cellular response to a certain re-occurring condition and therefore optimizes adaptation and fitness in a given environment. We found that genes coding for sterigmatocystin (ST) production in Aspergillus nidulans are activated stronger when cells are repeatedly exposed to nutrient starvation, compared to cells that experience this condition for the first time. We studied possible underlying mechanisms and found that persistence of the transcription factor AflR as well as a chromatin-based mechanism through histone H3 lysine 4 dimethylation (H3K4me2) are mediating the memory process. Our data indicate that AflR can undergo activation-inactivation cycles. On the other hand, an unknown mechanism mediated by a so far non-identified signal that originates from culture supernatants that were already starved once, specifically enhances the activation of starvation-dependent BGCs, including ST cluster activation. Finally, our ChIP-Seq data show that the ST cluster harbors i) three loci of constitutive and two of inducible H3K4me2 near the aflR locus, the latter two building up H3K4me3 after sufficient time, ii) bindings sites of histone demethylase KdmB near these H3K4me2 marks. Overall, using the ST cluster as model we provide insights on transcriptional memory and BGC activation dynamics in A. nidulans. A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) and then shifted on Salt medium (no C or N source) for 4 hours --> AMM_20h_Salt_4h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) --> AMM_24h A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) , shifted on Salt medium (no C or N source) for 4 hours , shifted on AMM for 2 h --> AMM_20h_Salt_4h_AMM_2h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) , shifted on AMM for 2 h --> AMM_24h_AMM_2h A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) , shifted on Salt medium (no C or N source) for 4 hours , shifted on AMM for 2 h, shifted on Salt medium (no C or N source) for 1 hour --> AMM_20h_Salt_4h_AMM_2h_Salt_1h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) , shifted on AMM for 2 h, shifted on Salt medium (no C or N source) for 1 hour --> AMM_24h_AMM_2h_Salt_1h A. nidulans was growth for 20h on Aspergillus minimal medium (AMM) , shifted on Salt medium (no C or N source) for 4 hours , shifted on AMM for 2 h, shifted on used medium (C or N source used up by A. nidulans growth for 48h) for 1 hour --> AMM_20h_Salt_4h_AMM_2h_Old_1h A. nidulans was growth for 24h on Aspergillus minimal medium (AMM) , shifted on AMM for 2 h, shifted on used medium (C or N source used up by A. nidulans growth for 48h) for 1 hour --> AMM_24h_AMM_2h_Old_1h A nd B denote biological replicates, 1 and 2 technical replicates

生物体频繁暴露于波动的环境与营养条件中。转录记忆（transcriptional memory）已被证实是应对此类波动的核心调控机制：其可提升细胞对重复出现的特定条件的响应速度与响应强度，进而优化生物体在特定环境中的适应性与适合度。本研究发现，相较于首次经历营养饥饿的构巢曲霉（Aspergillus nidulans）细胞，经反复暴露于营养饥饿条件的细胞中，负责合成粪壳菌素（sterigmatocystin, ST）的基因激活程度显著更强。我们对潜在的调控机制展开探究后发现，转录因子AflR的持续留存，以及基于染色质的组蛋白H3赖氨酸4二甲基化（histone H3 lysine 4 dimethylation, H3K4me2）调控通路，共同介导了该记忆过程。我们的实验数据显示，AflR可经历激活-失活循环。此外，一种由此前未被鉴定的信号介导的未知调控机制——该信号源自已经历过一轮饥饿的培养上清液——可特异性增强依赖营养饥饿的生物合成基因簇（biosynthetic gene cluster, BGC）的激活，其中便包含ST基因簇的激活。最后，我们的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-Seq）数据显示，ST基因簇具备以下特征：i) 在aflR基因座附近存在3个组成型H3K4me2位点与2个诱导型H3K4me2位点，其中后2个位点可在培养足够时长后形成H3K4me3；ii) 在上述H3K4me2标记区域附近存在组蛋白去甲基化酶KdmB的结合位点。综上，本研究以ST基因簇为模型，为解析构巢曲霉（Aspergillus nidulans）中的转录记忆与BGC激活动态机制提供了全新见解。
以下为各组实验样本的培养条件与标识：
1. 构巢曲霉在曲霉基础培养基（Aspergillus minimal medium, AMM）中培养20小时，随后转移至不含碳源与氮源的盐培养基中培养4小时，样本标识为AMM_20h_Salt_4h
2. 构巢曲霉在AMM中培养24小时，样本标识为AMM_24h
3. 构巢曲霉在AMM中培养20小时，转移至不含碳源与氮源的盐培养基培养4小时，再转回AMM中培养2小时，样本标识为AMM_20h_Salt_4h_AMM_2h
4. 构巢曲霉在AMM中培养24小时，转回AMM中培养2小时，样本标识为AMM_24h_AMM_2h
5. 构巢曲霉在AMM中培养20小时，转移至不含碳源与氮源的盐培养基培养4小时，转回AMM培养2小时，再次转移至不含碳源与氮源的盐培养基培养1小时，样本标识为AMM_20h_Salt_4h_AMM_2h_Salt_1h
6. 构巢曲霉在AMM中培养24小时，转回AMM培养2小时，再次转移至不含碳源与氮源的盐培养基培养1小时，样本标识为AMM_24h_AMM_2h_Salt_1h
7. 构巢曲霉在AMM中培养20小时，转移至不含碳源与氮源的盐培养基培养4小时，转回AMM培养2小时，随后转移至经48小时构巢曲霉生长消耗殆尽碳源与氮源的旧培养基中培养1小时，样本标识为AMM_20h_Salt_4h_AMM_2h_Old_1h
8. 构巢曲霉在AMM中培养24小时，转回AMM培养2小时，随后转移至经48小时构巢曲霉生长消耗殆尽碳源与氮源的旧培养基中培养1小时，样本标识为AMM_24h_AMM_2h_Old_1h
注：A与B代表生物学重复，1与2代表技术重复