Identification of G1-Regulated Genes in Normally Cycling Human Cells

Abstract

BackgroundObtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked.Methodology and FindingsWe used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb) by qPCR to further validate the newly identified genes.Conclusion and SignificanceGenome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

Meta-analysis reveals conserved cell cycle transcriptional network across multiple human cell types

“…However, G1-associated gene expression remains largely elusive. For example, an additional study specifically aimed to identify genes differentially expressed during G 1 phase in cycling HeLa cells [44] identified 200 transcripts which however did not match any of those found in the four studies nor did they cluster together in our analyses. Further analyses focused on characterizing the G 1 phase transcriptional regulation will be then of value, especially in view of its crucial role in aberrant proliferation.…”

A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition

“…Given these uncertainties in the gene regulatory milieu at the mitosis-G1 transition, might there be altered transcriptional output during this cell cycle phase? A microarraybased study identified approximately 200 mature mRNAs that fluctuate during early G1 in mammalian cells (Beyrouthy et al 2008), but it is unknown to what extent changes in transcriptional activity, versus post-transcriptional modulation, are responsible for these fluctuations. Several studies have directly quantified transcriptional activity over time in cells transitioning from mitosis to interphase (Blobel et al 2009;Anup Dey et al 2009;Muramoto et al 2010;Zhao et al 2011;Fukuoka et al 2012;Caravaca et al 2013), using RT-qPCR of primary transcripts of candidate genes (Blobel et al 2009;Anup Dey et al 2009;Fukuoka et al 2012;Caravaca et al 2013), live-cell imaging of transcription of act-5 in Dictyostelium (Muramoto et al 2010) and a multi-copy reporter locus in a human cell line (Zhao et al 2011), and microarray-based measurements of nascent transcripts (Fukuoka et al 2012).…”

Transcription profiling during the cell cycle shows that a subset of Polycomb-targeted genes is upregulated during DNA replication

“…Consequently, more cycling genes can be detected in a highly synchronous culture than in a culture where at most 50% of the cells are synchronized. Moreover, as the only human cell line—in addition to primary fibroblasts (1,3,4)—profiled for cell cycle expression so far is the cervical cancer cell line HeLa (2,5), it is unclear to what extent cell type-specific factors affect reported differences in cycling genes.…”