Yeast heterozygous FHL1 deletion mutant. Saccharomyces cerevisiae

Forkhead transcription factor FHL1 gene was required for replicative lifespan as well as cell proliferation in yeast. In this study, to see how Fhl1p determines the lifespan, we performed a DNA microarray analysis of heterozygous diploid strain deleted for FHL1 and, from the Fhl1p-target genes, screened for a lifespan-related gene. Transcriptomic profiles showed large increases and moderate decreases in transcripts in the fhl1/FHL1 mutant. Pathway analysis suggested upregulation of most of carbon metabolisms (glycolysis, gluconeogenesis, and TCA cycle) and downregulation of translation (ribosomal proteins and translation factors) in the FHL1-deficient mutant. We found a remarkable downregulation of a set of phosphate metabolism genes and ribonucleotide reductase large subunit genes, RNR1 and RNR3. We demonstrated that Fhl1p regulates RNR1 gene transcription to maintain dNTP levels, modulating longevity against replication stress and genomic instability. Overall design: Total RNA samples were prepared from wild-type BY4743 and its isogenic fhl1/FHL1 mutant cells grown in YPD medium at 30°C to OD600 of 1.0. Antisense RNA was synthesized and labeled with Cy3 for the fhl1/FHL1 mutant and with Cy5 for the wild type. Each mixture of Cy3- and Cy5-labeled antisense RNA was hybridized on a 3D-Gene Yeast Oligo Chip 6K (Toray, Kanagawa, Japan). The hybridized array was scanned with a 3D-Gene Scanner 3000 (Toray).

叉头框转录因子FHL1（Forkhead transcription factor FHL1）基因是酵母复制寿命与细胞增殖所必需的。本研究为阐明Fhl1蛋白（Fhl1p）如何调控酵母复制寿命，对敲除FHL1的异源二倍体菌株开展了DNA微阵列分析，并从Fhl1蛋白的靶基因中筛选寿命相关调控基因。转录组谱结果显示，fhl1/FHL1杂合突变体的转录本存在显著上调与轻度下调的表达变化。通路富集分析表明，FHL1缺陷突变体中多数碳代谢通路（糖酵解、糖异生及三羧酸循环）呈现上调趋势，而翻译相关通路（核糖体蛋白与翻译因子）则呈现下调趋势。本研究发现一组磷酸代谢基因与核糖核苷酸还原酶大亚基基因RNR1、RNR3均出现显著下调。实验证实，Fhl1蛋白可通过调控RNR1基因的转录以维持脱氧核苷三磷酸（dNTP）水平，进而在复制压力与基因组不稳定的条件下调控酵母寿命。
实验整体设计：将野生型BY4743菌株及其同基因fhl1/FHL1突变体菌株接种于YPD培养基中，于30℃培养至OD₆₀₀为1.0，随后提取总RNA样本；分别合成反义RNA，以Cy3标记fhl1/FHL1突变体样本的反义RNA，以Cy5标记野生型样本的反义RNA；将两种荧光标记的反义RNA混合后，在3D-Gene酵母寡核苷酸芯片6K（日本神奈川东丽公司）上进行杂交；杂交完成后的芯片采用3D-Gene Scanner 3000（东丽公司）进行扫描。