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Type: Book chapter
Title: Characterization of Mammalian Regulatory Complexes at Single-Locus Resolution Using TINC
Author: Knaupp, A.S.
Schittenhelm, R.B.
Polo, J.M.
Citation: Chromatin: Methods and Protocols, 2022 / Horsfield, J., Marsman, J. (ed./s), vol.2458, Ch.10, pp.175-193
Publisher: Humana
Publisher Place: New York, NY
Issue Date: 2022
Series/Report no.: Methods in Molecular Biology; 2458
ISBN: 9781071621394
Editor: Horsfield, J.
Marsman, J.
Statement of
Anja S. Knaupp, Ralf B. Schittenhelm, and Jose M. Polo
Abstract: In mammalian cells, multiprotein complexes form at specific genomic regulatory elements (REs) to control gene expression, which in turn is ultimately responsible for cellular identity. Consequently, insight into the molecular composition of these regulatory complexes is of major importance for our understanding of any physiological or pathological cellular state or transition. However, it remains extremely difficult to identify the protein complex(es) assembled at a specific RE in the mammalian genome using conventional approaches. We therefore developed a novel single locus isolation technique based on Transcription Activator-Like Effector (TALE) proteins termed TALE-mediated isolation of nuclear chromatin (TINC). When coupled with high-resolution mass spectrometry, TINC enables the identification and characterization of protein complexes formed at any RE of interest. Using the Nanog promoter in mouse embryonic stem cells as proof of concept, this chapter describes in detail the novel TINC methodology as well as subsequent mass spectrometric considerations.
Keywords: Single-locus pulldown
Regulatory complex
Transcriptional regulation
Affinity purification
Transcription activator-like effector proteins
Chromatin immuno-precipitation
Mass spectrometry
Description: Book series - Print ISSN: 1064-3745 ; Electronic ISSN: 1940-6029
Rights: © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
DOI: 10.1007/978-1-0716-2140-0_10
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Appears in Collections:Medicine publications
South Australian Immunogenomics Cancer Institute (SAIGENCI) publications

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