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 Ccnb...

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Autores principales:	Nidhi Vishnoi, Jie Yao
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4ef03e3955a142ac8db814f6ff699253
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spelling	oai:doaj.org-article:4ef03e3955a142ac8db814f6ff6992532021-12-02T12:30:44ZSingle-cell, single-mRNA analysis of Ccnb1 promoter regulation10.1038/s41598-017-02240-y2045-2322https://doaj.org/article/4ef03e3955a142ac8db814f6ff6992532017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02240-yhttps://doaj.org/toc/2045-2322Abstract 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.Nidhi VishnoiJie YaoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-15 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nidhi Vishnoi Jie Yao Single-cell, single-mRNA analysis of Ccnb1 promoter regulation
description	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.
format	article
author	Nidhi Vishnoi Jie Yao
author_facet	Nidhi Vishnoi Jie Yao
author_sort	Nidhi Vishnoi
title	Single-cell, single-mRNA analysis of Ccnb1 promoter regulation
title_short	Single-cell, single-mRNA analysis of Ccnb1 promoter regulation
title_full	Single-cell, single-mRNA analysis of Ccnb1 promoter regulation
title_fullStr	Single-cell, single-mRNA analysis of Ccnb1 promoter regulation
title_full_unstemmed	Single-cell, single-mRNA analysis of Ccnb1 promoter regulation
title_sort	single-cell, single-mrna analysis of ccnb1 promoter regulation
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/4ef03e3955a142ac8db814f6ff699253
work_keys_str_mv	AT nidhivishnoi singlecellsinglemrnaanalysisofccnb1promoterregulation AT jieyao singlecellsinglemrnaanalysisofccnb1promoterregulation
_version_	1718394333683515392

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

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