Mu X, Spanos S A, Shiloach J, Kimmel A
Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892-8028, USA.
Development. 2001 Jul;128(13):2569-79. doi: 10.1242/dev.128.13.2569.
During aggregation, Dictyostelium establish nanomolar oscillation waves of extracellular cAMP, but as development progresses, cells become responsive to higher, non-fluctuating concentrations of cAMP. The regulation of the promoter responsible for expression of cAMP receptor subtype 1, CAR1, during aggregation reflects these signaling variations. Transcription of CAR1 from the early, aggregation promoter is activated by cAMP pulsing, but is repressed by continuous exposure to micromolar concentrations of cAMP. Deletion and mutation analyses of this promoter had defined an element essential for cAMP-regulated expression, and mobility shift assay, DNA crosslinking and DNase I footprinting experiments had identified a nuclear protein (CRTF) with zinc-dependent sequence binding specificity. In our study, CRTF was purified to homogeneity, peptides were sequenced and full-length cDNAs were obtained. The deduced CRTF protein is approximately 100 kDa with a C-terminal, zinc finger-like motif required for DNA binding; CRTF purified from cells, however, represents only a 40 kDa C-terminal fragment that retains DNA-binding activity. As might have been predicted if CRTF were essential for the regulation of CAR1, crtf-null strains fail to develop under standard conditions or to exhibit induced expression of CAR1 or other cAMP-regulated genes. Furthermore, crtf-nulls also fail to sporulate, even under conditions that bypass the dependence on early cAMP signaling pathways. In addition, early developmental events of crtf-null strains could be rescued with exogenous cAMP treatment, constitutive expression of CAR1 or co-development with wild-type cells; however, these treatments were insufficient to promote sporulation. This suggests a cell-autonomous role for CRTF during late development that is separate from its capacity to control CAR1 expression. Finally, ablation of CRTF promotes a precocious induction of certain cAMP-dependent gene expression pathways. We suggest that CRTF may function to help insulate distinct pathways from simultaneous and universal activation by cAMP. CRTF, thus, exhibits multiple complex and independent regulatory functions during Dictyostelium development.
在聚集过程中，盘基网柄菌会建立细胞外cAMP的纳摩尔振荡波，但随着发育的进行，细胞会对更高的、非波动浓度的cAMP产生反应。负责cAMP受体亚型1（CAR1）表达的启动子在聚集过程中的调控反映了这些信号变化。在聚集早期启动子上，CAR1的转录由cAMP脉冲激活，但在持续暴露于微摩尔浓度的cAMP时会受到抑制。对该启动子的缺失和突变分析确定了一个对cAMP调节表达至关重要的元件，迁移率变动分析、DNA交联和DNase I足迹实验鉴定出一种具有锌依赖性序列结合特异性的核蛋白（CRTF）。在我们的研究中，CRTF被纯化至同质，对肽段进行了测序并获得了全长cDNA。推导的CRTF蛋白约为100 kDa，具有DNA结合所需的C末端锌指样基序；然而，从细胞中纯化的CRTF仅代表一个保留DNA结合活性的40 kDa C末端片段。如果CRTF对CAR1的调节至关重要，那么正如所预测的那样，crtl基因缺失的菌株在标准条件下无法发育，也无法表现出CAR1或其他cAMP调节基因的诱导表达。此外，即使在绕过对早期cAMP信号通路依赖的条件下，crtl基因缺失的菌株也无法形成孢子。此外，用外源性cAMP处理、CAR1的组成型表达或与野生型细胞共同发育可以挽救crtl基因缺失菌株的早期发育事件；然而，这些处理不足以促进孢子形成。这表明CRTF在发育后期具有细胞自主作用，这与其控制CAR1表达的能力是分开的。最后，CRTF的缺失促进了某些cAMP依赖性基因表达途径的早熟诱导。我们认为CRTF可能起到帮助将不同途径与cAMP的同时普遍激活隔离开来的作用。因此，CRTF在盘基网柄菌发育过程中表现出多种复杂且独立的调节功能。
