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Single-cell, single-mRNA analysis of Ccnb1 promoter regulation.

Abstract：

:Promoter activation drives gene transcriptional output. Here we report generating site-specifically integrated single-copy promoter transgenes and measuring their expression to indicate promoter activities at single-mRNA level. mRNA counts, Pol II density and Pol II firing rates of the Ccnb1 promoter transgene resembled those of the native Ccnb1 gene both among asynchronous cells and during the cell cycle. We observed distinct activation states of the Ccnb1 promoter among G1 and G2/M cells, suggesting cell cycle-independent origin of cell-to-cell variation in Ccnb1 promoter activation. Expressing a dominant-negative mutant of NF-YA, a key transcriptional activator of the Ccnb1 promoter, increased its "OFF"/"ON" time ratios but did not alter Pol II firing rates during the "ON" period. Furthermore, comparing H3K4me2 and H3K79me2 levels at the Ccnb1 promoter transgene and the native Ccnb1 gene indicated that the enrichment of these two active histone marks did not predispose higher transcriptional activities. In summary, this experimental system enables bridging transcription imaging with molecular analysis to provide novel insights into eukaryotic transcriptional regulation.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Vishnoi N,Yao J

doi

10.1038/s41598-017-02240-y

subject

Has Abstract

pub_date

2017-05-18 00:00:00

pages

2065

issue

1

issn

2045-2322

pii

10.1038/s41598-017-02240-y

journal_volume

7

pub_type

杂志文章

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