HSF1-expression: Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/TATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TBP recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class. The genotype of yeast strains was modified for nuclear depletion experiments as follows: By4742-aa : tor1-1; fpr1del; RPL13A-FKBP12-NAT;MET15;his3-1;leu2; lys2; ura3; matalpha. Hsf1-aa: Hsf1-FRB-yEGFP. Hsf1 was depleted from the nucleus by adding either 7.5 uM (standard) or 0.15 uM (slow) rapamycin to the cells. Newly synthesized mRNA were extracted using 4tU labeling at different time points: before (0 min) and 15, 30, 60 and 90 minutes after Hsf1 depletion. mRNA synthesis rates were measured using two-channel microarrays. Two independent cultures were hybridized on two separate microarrays. For the first hybridization, the Cy5 (red) labeled cRNA from the WT-aa or Hsf1-aa strain is hybridized together with the Cy3 (green) labeled cRNA from a (4tU-)refpool. This 4tU-refpool was created by pooling 4tU labeled mRNA from two independently grown WT-aa strains without addition of rapamycin. For the replicate hybridization of the slow depletion timecourse, the labels are swapped.

组成型表达的持家基因（housekeeping genes）与调控基因之间的核心差异常被重点区分。尽管二者通常以不同的DNA元件、染色质架构及辅因子为特征，但目前尚不清楚启动子（promoter）类别在多大程度上严格遵循可调控性规则，以及何种分子机制决定了这类差异。我们的研究表明，相较于依赖同一激活因子Hsf1的TFIID/TATA样启动子（TATA-like promoters），以SAGA主导的TATA框（TATA-box）启动子对激活因子剂量变化的响应更为显著。因此，可调控性是启动子类别的固有属性。进一步分析显示，SAGA/TATA框启动子的动态性更强，因为通过SAGA招募的TATA框结合蛋白（TATA-binding protein, TBP）易被Mot1移除。此外，在激活因子缺失后，SAGA/TATA框启动子区域的核小体构型会发生移位，且似乎更不利于预起始复合物（preinitiation complex, PIC）的形成。本研究结果阐释了持家基因与调控基因之间的根本差异，同时揭示了组合调控的另一维度：激活因子可根据核心启动子（core promoter）类别的不同，引发不同的响应效果。为开展细胞核缺失实验，我们对酵母菌株的基因型进行了如下修饰：By4742-aa：tor1-1；fpr1Δ；RPL13A-FKBP12-NAT；MET15；his3-1；leu2；lys2；ura3；matalpha。Hsf1-aa：Hsf1-FRB-yEGFP。通过向细胞中添加7.5 μM（标准剂量）或0.15 μM（慢速剂量）的雷帕霉素（rapamycin），可使Hsf1从细胞核中被清除。我们在不同时间点（Hsf1缺失前即0分钟，以及缺失后15、30、60、90分钟）采用4tU标记法提取新合成的信使RNA（messenger RNA, mRNA）。采用双通道微阵列技术测定mRNA的合成速率。将两份独立培养的样本分别在两张独立的微阵列上进行杂交。在首次杂交中，将来自WT-aa或Hsf1-aa菌株的Cy5（红色）标记的互补RNA（complementary RNA, cRNA），与来自（4tU-）参考池（refpool）的Cy3（绿色）标记的cRNA进行共杂交。该4tU参考池是通过将两份未添加雷帕霉素、独立培养的WT-aa菌株的4tU标记mRNA混合制备得到的。在慢速缺失时间序列的重复杂交实验中，荧光标记的通道进行了互换。