Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon, Korea.
PLoS One. 2012;7(8):e42409. doi: 10.1371/journal.pone.0042409. Epub 2012 Aug 10.
Gaf1 is the first GATA family zinc-finger transcription factor identified in Schizosaccharomyces pombe. Here, we report that Gaf1 functions as a negatively acting transcription factor of ste11(+), delaying the entrance of cells exposed to transient nitrogen starvation into the meiotic cycle. gaf1Δ strains exhibited accelerated G(1)-arrest upon nitrogen starvation. Moreover, gaf1Δ mutation caused increased mating and sporulation frequency under both nitrogen-starved and unstarved conditions, while overexpression of gaf1(+) led to a significant impairment of sporulation. By microarray analysis, we found that approximately 63% (116 genes) of the 183 genes up-regulated in unstarved gaf1Δ cells were nitrogen starvation-responsive genes, and furthermore that 25 genes among the genes up-regulated by gaf1Δ mutation are Ste11 targets (e.g., gpa1(+), ste4(+), spk1(+), ste11(+), and mei2(+)). The phenotype caused by gaf1Δ mutation was masked by ste11Δ mutation, indicating that ste11(+) is epistatic to gaf1(+) with respect to sporulation efficiency, and accordingly that gaf1(+) functions upstream of ste11(+) in the signaling pathway governing sexual development. gaf1Δ strains showed accelerated ste11(+) expression under nitrogen starvation and increased ste11(+) expression even under normal conditions. Electrophoretic mobility shift assay analysis demonstrated that Gaf1 specifically binds to the canonical GATA motif (5'-HGATAR-3') spanning from -371 to -366 in ste11(+) promoter. Consequently, Gaf1 provides the prime example for negative regulation of ste11(+) transcription through direct binding to a cis-acting motif of its promoter.
Gaf1 是第一个在裂殖酵母中发现的 GATA 家族锌指转录因子。在这里，我们报告说 Gaf1 作为 ste11(+)的负调控转录因子发挥作用，延迟暴露于短暂氮饥饿的细胞进入减数分裂周期。gaf1Δ 菌株在氮饥饿时表现出加速的 G1 期阻滞。此外，gaf1Δ 突变导致在氮饥饿和未饥饿条件下交配和孢子形成频率增加，而 gaf1(+)的过表达导致孢子形成显著受损。通过微阵列分析，我们发现，在未饥饿的 gaf1Δ 细胞中上调的 183 个基因中，约 63%（116 个基因）是氮饥饿响应基因，而且 gaf1Δ 突变上调的基因中有 25 个是 Ste11 靶基因（例如，gpa1(+)、ste4(+)、spk1(+)、ste11(+)和 mei2(+)）。gaf1Δ 突变引起的表型被 ste11Δ 突变掩盖，表明 ste11(+)在孢子形成效率上对 gaf1(+)是上位的，因此 gaf1(+)在调控有性发育的信号通路中位于 ste11(+)的上游。gaf1Δ 菌株在氮饥饿下表现出加速的 ste11(+)表达，甚至在正常条件下也表现出增加的 ste11(+)表达。电泳迁移率变动分析表明，Gaf1 特异性结合 ste11(+)启动子中跨越-371 到-366 的典型 GATA 基序（5'-HGATAR-3'）。因此，Gaf1 通过直接结合其启动子的顺式作用元件，为 ste11(+)转录的负调控提供了范例。
