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