Characterization of Mammalian Regulatory Complexes at Single-Locus Resolution Using TINC

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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.

Original languageEnglish
Title of host publicationChromatin
Subtitle of host publicationMethods and Protocols
EditorsJulia Horsfield, Judith Marsman
Place of PublicationNew York NY USA
PublisherHumana Press
Chapter10
Pages175-193
Number of pages19
ISBN (Electronic)9781071621400
ISBN (Print)9781071621394
DOIs
Publication statusPublished - 2022

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume2458
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Affinity purification
  • Chromatin immuno-precipitation
  • Epigenetics
  • Mass spectrometry
  • Proteomics
  • Regulatory complex
  • Single-locus pulldown
  • Transcription activator-like effector proteins
  • Transcriptional regulation

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