Pilot RNAi screening using mammalian cell-based system identifies novel putative silencing factors including Kat5/Tip60

Anuroop Venkateswaran Venkatasubramani; Katy McLaughlin; Giovanny Rodriguez Blanco; Vladimir Larionov; Alexander Kagansky; Anuroop Venkateswaran Venkatasubramani; Katy McLaughlin; Giovanny Rodriguez Blanco; Vladimir Larionov; Alexander Kagansky

doi:10.3934/biophy.2015.4.570

AIMS Biophysics

2015, Volume 2, Issue 4: 570-584. doi: 10.3934/biophy.2015.4.570

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Pilot RNAi screening using mammalian cell-based system identifies novel putative silencing factors including Kat5/Tip60

Anuroop Venkateswaran Venkatasubramani ^1,2,
Katy McLaughlin ¹,
Giovanny Rodriguez Blanco ¹,
Vladimir Larionov ³,
Alexander Kagansky ^{1
,
,}

1.
Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom;
2.
Department of Biosciences, P.O. Box 65 (Viikkinkaari 1), 00014, University of Helsinki, Finland;
3.
Center for Cancer Research, National Cancer Institute, Building 37, Room 5032A, Bethesda, MD 20892, USA

Received: 21 August 2015 Accepted: 14 October 2015 Published: 20 October 2015

Epigenetic silencing is the reversible inactivation of a gene transcription which occurs as a result of changes in the structure of the chromatin that can be successfully inherited from parent to daughter cells. It involves non-genetic mutations within the genome, as well as post-transcriptional and post-translational mechanisms. Existence of these mechanisms at various levels warrants their role in development and disease and thus it is crucial to study different factors and mechanisms of silencing. The aim of our study was to establish a method for rapid screening for the loss of epigenetic silencing in mammalian cells, to identify factor(s) involved in epigenetic silencing, and to get insights into their mechanism of action. For this purpose, we used RNAi screening approach using shRNAs that targeted our genes of interest. We employed a modified mouse cell line which contained a GFP transgene under the control of CMV promoter which has been silenced by epigenetic modifications. Our screening identified several proteins as epigenetic silencing regulators including Kat5/Tip60, an acetyltransferase of MYST family of proteins. To characterize its function, we performed preliminary experiments using microscopy and Western blot analysis of histone marks. We observed changes in H4 acetylation levels in Kat5/Tip60 knockdown cells. Our study thus serves as a pilot for a genome-wide silencing screening using mammalian cells, and provides preliminary results suggesting that Kat5 can be considered as a silencing factor, which, we propose, could function by maintaining H4 acetylation patterns.
- RNAi screening,
- acetyltransferase,
- Kat5/Tip60,
- silencing,
- epigenetics,
- chromatin,
- histone,
- acetylation
Citation: Anuroop Venkateswaran Venkatasubramani, Katy McLaughlin, Giovanny Rodriguez Blanco, Vladimir Larionov, Alexander Kagansky. Pilot RNAi screening using mammalian cell-based system identifies novel putative silencing factors including Kat5/Tip60[J]. AIMS Biophysics, 2015, 2(4): 570-584. doi: 10.3934/biophy.2015.4.570

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Abstract

Epigenetic silencing is the reversible inactivation of a gene transcription which occurs as a result of changes in the structure of the chromatin that can be successfully inherited from parent to daughter cells. It involves non-genetic mutations within the genome, as well as post-transcriptional and post-translational mechanisms. Existence of these mechanisms at various levels warrants their role in development and disease and thus it is crucial to study different factors and mechanisms of silencing. The aim of our study was to establish a method for rapid screening for the loss of epigenetic silencing in mammalian cells, to identify factor(s) involved in epigenetic silencing, and to get insights into their mechanism of action. For this purpose, we used RNAi screening approach using shRNAs that targeted our genes of interest. We employed a modified mouse cell line which contained a GFP transgene under the control of CMV promoter which has been silenced by epigenetic modifications. Our screening identified several proteins as epigenetic silencing regulators including Kat5/Tip60, an acetyltransferase of MYST family of proteins. To characterize its function, we performed preliminary experiments using microscopy and Western blot analysis of histone marks. We observed changes in H4 acetylation levels in Kat5/Tip60 knockdown cells. Our study thus serves as a pilot for a genome-wide silencing screening using mammalian cells, and provides preliminary results suggesting that Kat5 can be considered as a silencing factor, which, we propose, could function by maintaining H4 acetylation patterns.

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