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Abstract：
Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3\'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3\'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3\'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3\'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.
摘要：
肌醇焦磷酸盐是调节真核生物类群中细胞磷酸盐稳态的信号分子。在裂殖酵母中，其中磷酸盐调节子（包含磷酸盐获取基因pho1，pho84和tgp1）在磷酸盐充足的条件下被lncRNA介导的转录干扰抑制，增加IP8水平的肌醇焦磷酸酶的突变通过引起lncRNA转录的早熟终止来抑制PHO调节子。由于甘油磷酸二酯转运蛋白Tgp1的过表达，导致IP8过多的Asp1焦磷酸酶突变在YES培养基中具有细胞毒性。IP8中毒通过裂解/聚腺苷酸化和终止因子的突变得到改善，Pol2CTD码的扰动，以及充当肌醇焦磷酸传感器的SPX结构域蛋白中的突变。这里,我们表明，IP8毒性通过SNF22+的缺失得到缓解，编码SWI/SNF染色质重塑复合物的ATPase亚基的基因，通过ATPase失活snf22-（D996A-E997A）等位基因，并通过缺失编码SWI/SNF亚基Sol1的基因。在磷酸盐充足的细胞中，snf22过度抑制pho1表达的缺失；抑制Pol2CTD突变引起的pho1抑制，终止因子Seb1,Asp1焦磷酸酶,和14-3-3蛋白Rad24（有利于早熟的prtlncRNA终止）；并在磷酸盐饥饿期间延迟pho1诱导。RNA分析和缺乏突变协同作用表明Snf22不影响3'-加工/终止。使用报告分析，我们发现Snf22对tgp1和pho1启动子的活性很重要，但不是驱动PHO抑制lncRNAs合成的启动子。snf22Δ和snf22-（D996A-E997A）细胞的转录谱鉴定出另外一组66个蛋白质编码基因，这些基因在两个突变体中均下调。IMPORTANCElncRNA介导的干扰对裂变酵母PHO基因tgp1，pho1和pho84的抑制对肌醇焦磷酸动力学敏感。细胞毒性asp1-STF等位基因通过IP8作为早熟lncRNA3'-加工/终止的激动剂的作用抑制PHO基因。Pol2CTD和3'-加工/终止机制的突变减轻了IP8中毒，从而减轻了毒性IP8水平对终止的影响。在这项研究中,正向遗传筛选显示,SWI/SNF染色质重塑复合物的Snf22和Sol1亚基的突变抑制了IP8毒性.遗传和生化证据表明，SWI/SNF不影响3'-加工/终止或lncRNA启动子活性。相反，SWI/SNF对于激发PHOmRNA启动子至关重要。我们的结果暗示SWI/SNF的ATP依赖性核小体重塑活性是必要的，以确保PHO激活转录因子Pho7完全进入其在PHOmRNA启动子中的结合位点。
