Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits

Abstract We report that epigenetic silencing causes the loss of function of multi-transcript unit constructs that are integrated using CRISPR-Cas9. Using a modular two color reporter system flanked by selection markers, we demonstrate that expression heterogeneity does not correlate with sequence al...

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Autores principales:	Jan Zimak, Zachary W. Wagoner, Nellie Nelson, Brooke Waechtler, Hana Schlosser, Morgan Kopecky, Jie Wu, Weian Zhao
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/99a5ea87ae6540f48bce4ae7dceb0557
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spelling	oai:doaj.org-article:99a5ea87ae6540f48bce4ae7dceb05572021-12-02T13:57:37ZEpigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits10.1038/s41598-021-81975-12045-2322https://doaj.org/article/99a5ea87ae6540f48bce4ae7dceb05572021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81975-1https://doaj.org/toc/2045-2322Abstract We report that epigenetic silencing causes the loss of function of multi-transcript unit constructs that are integrated using CRISPR-Cas9. Using a modular two color reporter system flanked by selection markers, we demonstrate that expression heterogeneity does not correlate with sequence alteration but instead correlates with chromosomal accessibility. We partially reverse this epigenetic silencing via small-molecule inhibitors of methylation and histone deacetylation. We then correlate each heterogeneously-expressing phenotype with its expected epigenetic state by employing ATAC-seq. The stability of each expression phenotype is reinforced by selective pressure, which indicates that ongoing epigenetic remodeling can occur for over one month after integration. Collectively, our data suggests that epigenetic silencing limits the utility of multi-transcript unit constructs that are integrated via double-strand repair pathways. Our research implies that mammalian synthetic biologists should consider localized epigenetic outcomes when designing complex genetic circuits.Jan ZimakZachary W. WagonerNellie NelsonBrooke WaechtlerHana SchlosserMorgan KopeckyJie WuWeian ZhaoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jan Zimak Zachary W. Wagoner Nellie Nelson Brooke Waechtler Hana Schlosser Morgan Kopecky Jie Wu Weian Zhao Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
description	Abstract We report that epigenetic silencing causes the loss of function of multi-transcript unit constructs that are integrated using CRISPR-Cas9. Using a modular two color reporter system flanked by selection markers, we demonstrate that expression heterogeneity does not correlate with sequence alteration but instead correlates with chromosomal accessibility. We partially reverse this epigenetic silencing via small-molecule inhibitors of methylation and histone deacetylation. We then correlate each heterogeneously-expressing phenotype with its expected epigenetic state by employing ATAC-seq. The stability of each expression phenotype is reinforced by selective pressure, which indicates that ongoing epigenetic remodeling can occur for over one month after integration. Collectively, our data suggests that epigenetic silencing limits the utility of multi-transcript unit constructs that are integrated via double-strand repair pathways. Our research implies that mammalian synthetic biologists should consider localized epigenetic outcomes when designing complex genetic circuits.
format	article
author	Jan Zimak Zachary W. Wagoner Nellie Nelson Brooke Waechtler Hana Schlosser Morgan Kopecky Jie Wu Weian Zhao
author_facet	Jan Zimak Zachary W. Wagoner Nellie Nelson Brooke Waechtler Hana Schlosser Morgan Kopecky Jie Wu Weian Zhao
author_sort	Jan Zimak
title	Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
title_short	Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
title_full	Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
title_fullStr	Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
title_full_unstemmed	Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
title_sort	epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/99a5ea87ae6540f48bce4ae7dceb0557
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_version_	1718392275192512512

Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits

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