Engineered mouse H1 promoter mutants with superior RNA polymerase III activity

Jiaying Wu; Yufei Zhou; Di Zhao; Ran Xu; Jienan Wang; Hong Lin; Zhiwen Ding; Yunzeng Zou

Biochemistry and Biophysics Reports (Sep 2024)

Engineered mouse H1 promoter mutants with superior RNA polymerase III activity

Jiaying Wu,
Yufei Zhou,
Di Zhao,
Ran Xu,
Jienan Wang,
Hong Lin,
Zhiwen Ding,
Yunzeng Zou

Affiliations

Jiaying Wu: Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China
Yufei Zhou: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China
Di Zhao: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China
Ran Xu: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China; Shanghai Geriatric Medical Center, Shanghai, 201104, China
Jienan Wang: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China
Hong Lin: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China
Zhiwen Ding: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China; Departments of Cardiology, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai, 218120, China; Corresponding author. Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Yunzeng Zou: Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China; Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai, 200032, China; Departments of Cardiology, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai, 218120, China; Corresponding author. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.

Journal volume & issue: Vol. 39
p. 101795

Abstract

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Vectors incorporating the human H1 (hH1) promoter are being applied for RNA interference (RNAi) experiments and genome editing. Although extensive studies have been conducted on the hH1 promoter, our understanding of the mouse H1 promoter remains limited. In this study, we predicted the 163 bp mouse H1 (mH1) promoter and 84 bp mouse H1 core (mH1 core) promoter through global alignment and detected its RNA polymerase II (Pol II) and III activities through the expression of the EGFP and the abundance of artificial sequence, which were generally slightly weaker than those of the hH1 promoter. Furthermore, to boost its Pol III activity, we engineered various promoter mutants by introducing mutations or systematically swapping elements. Surprisingly, the Pol II activity of mH1 core mut5 with AT stretch was at least 2-fold greater than that of the wild type, making it a potential candidate for target protein expression purposes. Fortunately, the Pol III activities of mH1 mut1 and mH1 core mut5 were at least 1.5 times stronger than those of the parental promoters in human and mouse cell lines on account of AT stretch, as did the mH1 mut4 with AT stretch and proximal sequence element (PSE) and TATA box insertion mutations. We highly recommend these three promoters as valuable supplements to the type 3 Pol III promoter toolbox.

Published in Biochemistry and Biophysics Reports

ISSN: 2405-5808 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Science: Biology (General); Science: Chemistry: Organic chemistry: Biochemistry
Website: http://www.journals.elsevier.com/biochemistry-and-biophysics-reports/

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