Cell-cycle dynamics of nascent transcription and mature RNA accumulation are concordant in normal fibroblasts [RNA-seq]

Dynamic changes of gene expression is fundamental to cell cycle control. A prevailing hypothesis states that cell-cycle gene expression dynamics is driven by a widespread transcription-to-maturation lag, in which nascent transcription occurs in a cell-cycle phase preceding the phase with mature RNA accumulation, based on a study in cancer cells. Here, we test this paradigm in non-transformed human fibroblasts. We define mature RNAs and nascent transcription during the cell cycle using FUCCI reporters. The two dynamics are strongly concordant without a lag. Our data suggest a widespread transcription-to-maturation lag may be a feature of some cancer cells, but not of normal cells. Overall design: To investigate cell-cycle dynamics of gene expression in cycling non-transformed cells, we introduced the Fucci4 cell-cycle fluorescent reporters to the hTERT-immortalized human fibroblast cell line BJ-5ta (BJ-Fucci). We sorted BJ-Fucci cells into early G1 (mKO2–Clover–mTurquoise2–), G1 (mKO2+Clover–), early-S (mKO2+Clover+mTurquois+), and late-S/G2/M (mKO2–Clover+) phases using FACS. We sequenced poly A-selected RNAs using RNA-seq in these sorted populations. To define the nascent transcriptional dynamics, we sorted early G1, G1, and late-S/G2/M BJ-Fucci cells and performed GRO-seq, which quantifies nascent transcripts produced during a short incubation of permeabilized cells with bromouridine (BrU) by sequencing of BrU-incorporated RNAs.

基因表达的动态变化是细胞周期调控的核心基础。当前主流假说认为，细胞周期基因表达的动态性由广泛存在的转录至成熟滞后现象驱动：新生转录发生于成熟RNA积累所处细胞周期阶段的上游时相，该结论源自一项癌细胞研究。
本研究针对非转化型人成纤维细胞验证这一研究范式。我们借助FUCCI报告系统（FUCCI）对细胞周期中的成熟RNA与新生转录进行定义。二者的动态变化呈现高度一致性，且不存在转录滞后现象。
本研究数据表明，广泛存在的转录至成熟滞后现象或许是部分癌细胞的特征，而非正常细胞的共性。
实验整体设计：为探究增殖性非转化细胞中基因表达的细胞周期动态变化，我们将FUCCI4细胞周期荧光报告系统导入至经人端粒酶逆转录酶（hTERT）永生化的人成纤维细胞系BJ-5ta（以下简称BJ-Fucci）。
我们通过荧光激活细胞分选术（FACS）将BJ-Fucci细胞分为早G1期（mKO2–Clover–mTurquoise2–）、G1期（mKO2+Clover–）、早S期（mKO2+Clover+mTurquoise+）以及晚S/G2/M期（mKO2–Clover+）。针对上述分选得到的细胞群，我们采用RNA测序（RNA-seq）技术对聚腺苷酸（poly A）筛选后的RNA进行测序。
为明确新生转录的动态变化，我们分选了早G1期、G1期以及晚S/G2/M期的BJ-Fucci细胞，并开展了全局运行测序（GRO-seq）实验：该技术通过将透化细胞与溴尿苷（BrU）短时间共孵育，随后对掺入BrU的新生RNA进行测序，以此定量新生转录本的水平。