Transcription levels of a long noncoding RNA shape a cell fate regulatory circuit (TSS-seq)

Long noncoding RNAs (lncRNA) play diverse roles in gene regulation controlling key cellular processes, most notably, cell-fate programming {Anderson, 2016 #20;Flynn, 2014 #16;Guttman, 2011 #35;Wang, 2011 #18}. Many long noncoding RNAs (lncRNAs) act in cis through transcription-coupled chromatin alterations that drive changes in local gene expression { Martens, 2004 #47; Kim, 2012 #74;van Werven, 2012 #57;Hainer, 2011 #73;Kim, 2016 #41;Ard, 2016 #21;Latos, 2012 #7}. How transcription of some lncRNAs leads to activation of gene expression, while others inhibit and repress gene expression remains poorly understood {Kornienko, 2013 #17}. Here we investigated in S. cerevisiae the function of the lncRNA IRT2, which is expressed upstream in the promoter of the master regulator for entry into meiosis, IME1. We report the surprising finding that distinct levels of IRT2 transcription regulates opposing chromatin and transcription states in order to ensure that only diploids, and not haploids, enter meiosis and form gametes. In haploid cells, IRT2 transcription at very low levels is required for the correct induction of the adjacent lncRNA IRT1, which in turn represses the IME1 promoter and prevents meiotic entry {van Werven, 2012 #57}. Low levels of IRT2 transcription stimulates histone exchange delivering acetylated histone H3 lysine 56 (H3K56ac) to chromatin, thereby facilitating chromatin disassembly and recruitment of the transcriptional activator of IRT1, Rme1. Inhibiting IRT2 transcription, or mutations that resulted in cells lacking H3K56ac impairs Rme1 recruitment and IRT1 induction, and consequently haploid cells induce IME1 and undergo a lethal meiosis. In contrast to its function at low levels, increasing IRT2 transcription enhances transcription-coupled chromatin assembly and interferes with IRT1 expression, promoting IME1 expression and meiotic entry in diploid cells {Moretto, 2018 #10}. Thus, transcription of lncRNAs, even at very low levels, can play an important role in regulating gene expression, and changes in lncRNA transcription levels can confer distinct regulatory and cell fate outcomes. Overall design: 5' end RNA sequencing analysis of Saccharomyces cerevisiae SK1 strains. The SPO (low nutrient) condition correspond to strains grown in YPD to saturation, then shifted to pre-sporulation media for 16h before being transfered to sporulation media. the u6bsD mutant harbours a clean deletion (no marker) of the Ume6 binding site present 2306 bp upstream IME1 ATG. Two biological replicates of each condition were analysed. Details for library preparation are described below.

长链非编码RNA（long noncoding RNAs, lncRNA）在调控关键细胞过程的基因调控中发挥多样功能，其中尤以细胞命运编程最为显著{Anderson, 2016 #20;Flynn, 2014 #16;Guttman, 2011 #35;Wang, 2011 #18}。诸多长链非编码RNA可通过转录偶联的染色质改变以顺式（cis）方式发挥作用，进而改变局部基因表达{ Martens, 2004 #47; Kim, 2012 #74;van Werven, 2012 #57;Hainer, 2011 #73;Kim, 2016 #41;Ard, 2016 #21;Latos, 2012 #7}。部分长链非编码RNA的转录可激活基因表达，而另一些则会抑制乃至阻遏基因表达，其背后的机制仍不甚明晰{Kornienko, 2013 #17}。本研究针对酿酒酵母（Saccharomyces cerevisiae）中的长链非编码RNA IRT2展开功能探究，该RNA在减数分裂进入主调控因子IME1的启动子上游区域表达。我们发现了一项出人意料的现象：不同水平的IRT2转录可调控相反的染色质与转录状态，以此确保仅二倍体细胞（而非单倍体细胞）能够进入减数分裂并形成配子。在单倍体细胞中，低水平的IRT2转录是正确诱导邻近长链非编码RNA IRT1表达所必需的，而IRT1会进而阻遏IME1启动子，阻止减数分裂起始{van Werven, 2012 #57}。低水平IRT2转录可促进组蛋白交换，将乙酰化组蛋白H3赖氨酸56（H3K56ac）运送至染色质，从而推动染色质解离，并招募IRT1的转录激活因子Rme1。抑制IRT2转录，或是使细胞缺失H3K56ac的突变，均会损害Rme1的招募与IRT1的诱导，最终导致单倍体细胞异常激活IME1并发生致死性减数分裂。与低水平IRT2转录的功能相反，提高IRT2转录水平可增强转录偶联的染色质组装，抑制IRT1的表达，进而促进二倍体细胞中IME1的表达与减数分裂进入{Moretto, 2018 #10}。由此可见，即便处于极低水平，长链非编码RNA的转录也可在基因表达调控中发挥重要作用，而长链非编码RNA转录水平的变化可带来不同的调控效应与细胞命运结局。
实验整体设计：对酿酒酵母SK1菌株开展5'端RNA测序（5' end RNA sequencing）分析。SPO（低营养）培养条件指将菌株在YPD培养基中培养至饱和状态，随后转移至前孢子形成培养基培养16小时，再转接至孢子形成培养基。u6bsD突变体为在IME1 ATG起始密码子上游2306 bp处的Ume6结合位点进行了无痕敲除（无筛选标记）的菌株。每个培养条件均设置两个生物学重复进行分析。文库制备的详细步骤见下文。