HaloChIP-seq for Antibody-Independent Mapping of Mouse Transcription Factor Cistromes In Vivo

Ann Louise Hunter; Antony Adamson; Toryn Poolman; Magdalena Grudzien; Andrew Loudon; David Ray; David Bechtold

doi:10.21769/BioProtoc.4460

HaloChIP-seq for Antibody-Independent Mapping of Mouse Transcription Factor Cistromes In Vivo

Ann Louise Hunter, Antony Adamson, Toryn Poolman, Magdalena Grudzien, Andrew Loudon, David Ray, David Bechtold

Research output: Contribution to journal › Article › peer-review

Abstract

Chromatin immunoprecipitation (ChIP) maps, on a genome-wide scale, transcription factor binding sites, and the distribution of other chromatin-associated proteins and their modifications. As such, it provides valuable insights into mechanisms of gene regulation. However, successful ChIP experiments are dependent on the availability of a high-quality antibody against the target of interest. Using antibodies with poor sensitivity and specificity can yield misleading results. This can be partly circumvented by using epitope-tagged systems (e.g., HA, Myc, His), but these approaches are still antibody-dependent. HaloTag® is a modified dehalogenase enzyme, which covalently binds synthetic ligands. This system can be used for imaging and purification of HaloTag® fusion proteins, and has been used for ChIP in vitro. Here, we present a protocol for using the HaloTag® system for ChIP in vivo, to map, with sensitivity and specificity, the cistrome of a dynamic mouse transcription factor expressed at its endogenous locus.

Original language	English
Article number	e4460
Journal	Bio-protocol
Volume	12
Issue number	13
DOIs	https://doi.org/10.21769/BioProtoc.4460
Publication status	Published - 5 Jul 2022

Keywords

ChIP
Fusion protein
HaloTag®
NR1D1
Nuclear receptor
Tag

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10.21769/BioProtoc.4460

Cite this

@article{3a85a44b6b4e463d9cbbade22580f2e0,

title = "HaloChIP-seq for Antibody-Independent Mapping of Mouse Transcription Factor Cistromes In Vivo",

abstract = "Chromatin immunoprecipitation (ChIP) maps, on a genome-wide scale, transcription factor binding sites, and the distribution of other chromatin-associated proteins and their modifications. As such, it provides valuable insights into mechanisms of gene regulation. However, successful ChIP experiments are dependent on the availability of a high-quality antibody against the target of interest. Using antibodies with poor sensitivity and specificity can yield misleading results. This can be partly circumvented by using epitope-tagged systems (e.g., HA, Myc, His), but these approaches are still antibody-dependent. HaloTag{\textregistered} is a modified dehalogenase enzyme, which covalently binds synthetic ligands. This system can be used for imaging and purification of HaloTag{\textregistered} fusion proteins, and has been used for ChIP in vitro. Here, we present a protocol for using the HaloTag{\textregistered} system for ChIP in vivo, to map, with sensitivity and specificity, the cistrome of a dynamic mouse transcription factor expressed at its endogenous locus.",

keywords = "ChIP, Fusion protein, HaloTag{\textregistered}, NR1D1, Nuclear receptor, Tag",

author = "Hunter, {Ann Louise} and Antony Adamson and Toryn Poolman and Magdalena Grudzien and Andrew Loudon and David Ray and David Bechtold",

note = "Funding Information: This research was funded by the Biotechnology and Biological Sciences Research Council (BB/I018654/1 to D.A.B.), the Medical Research Council (Clinical Research Training Fellowship MR/N021479/1 to A.L.H.; MR/P00279X/1 to D.A.B.; MR/P011853/1 and MR/P023576/1 to D.W.R.), National Institute for Health Research Oxford Biomedical Research Centre, and the Wellcome Trust (107849/Z/15/Z, 107851/Z/15/Z). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This protocol was derived from two original research papers (Hunter et al., 2020, 2021), the Promega HaloCHIPTM manufacturer{\textquoteright}s instructions, and the Active Motif High Sensitivity Chromatin Prep kit protocol, with adaptations for adipose tissue work informed by the publications of other groups (Haim et al., 2013; Castellano-Castillo et al., 2018). We acknowledge the core facilities at the University of Manchester: the Genome Editing Unit, the Genomic Publisher Copyright: {\textcopyright} 2022 E-Century Publishing Corporation. All rights reserved.",

year = "2022",

month = jul,

day = "5",

doi = "10.21769/BioProtoc.4460",

language = "English",

volume = "12",

journal = "Bio-protocol",

issn = "2331-8325",

publisher = "Bio-protocol LLC",

number = "13",

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TY - JOUR

T1 - HaloChIP-seq for Antibody-Independent Mapping of Mouse Transcription Factor Cistromes In Vivo

AU - Hunter, Ann Louise

AU - Adamson, Antony

AU - Poolman, Toryn

AU - Grudzien, Magdalena

AU - Loudon, Andrew

AU - Ray, David

AU - Bechtold, David

N1 - Funding Information: This research was funded by the Biotechnology and Biological Sciences Research Council (BB/I018654/1 to D.A.B.), the Medical Research Council (Clinical Research Training Fellowship MR/N021479/1 to A.L.H.; MR/P00279X/1 to D.A.B.; MR/P011853/1 and MR/P023576/1 to D.W.R.), National Institute for Health Research Oxford Biomedical Research Centre, and the Wellcome Trust (107849/Z/15/Z, 107851/Z/15/Z). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This protocol was derived from two original research papers (Hunter et al., 2020, 2021), the Promega HaloCHIPTM manufacturer’s instructions, and the Active Motif High Sensitivity Chromatin Prep kit protocol, with adaptations for adipose tissue work informed by the publications of other groups (Haim et al., 2013; Castellano-Castillo et al., 2018). We acknowledge the core facilities at the University of Manchester: the Genome Editing Unit, the Genomic Publisher Copyright: © 2022 E-Century Publishing Corporation. All rights reserved.

PY - 2022/7/5

Y1 - 2022/7/5

N2 - Chromatin immunoprecipitation (ChIP) maps, on a genome-wide scale, transcription factor binding sites, and the distribution of other chromatin-associated proteins and their modifications. As such, it provides valuable insights into mechanisms of gene regulation. However, successful ChIP experiments are dependent on the availability of a high-quality antibody against the target of interest. Using antibodies with poor sensitivity and specificity can yield misleading results. This can be partly circumvented by using epitope-tagged systems (e.g., HA, Myc, His), but these approaches are still antibody-dependent. HaloTag® is a modified dehalogenase enzyme, which covalently binds synthetic ligands. This system can be used for imaging and purification of HaloTag® fusion proteins, and has been used for ChIP in vitro. Here, we present a protocol for using the HaloTag® system for ChIP in vivo, to map, with sensitivity and specificity, the cistrome of a dynamic mouse transcription factor expressed at its endogenous locus.

AB - Chromatin immunoprecipitation (ChIP) maps, on a genome-wide scale, transcription factor binding sites, and the distribution of other chromatin-associated proteins and their modifications. As such, it provides valuable insights into mechanisms of gene regulation. However, successful ChIP experiments are dependent on the availability of a high-quality antibody against the target of interest. Using antibodies with poor sensitivity and specificity can yield misleading results. This can be partly circumvented by using epitope-tagged systems (e.g., HA, Myc, His), but these approaches are still antibody-dependent. HaloTag® is a modified dehalogenase enzyme, which covalently binds synthetic ligands. This system can be used for imaging and purification of HaloTag® fusion proteins, and has been used for ChIP in vitro. Here, we present a protocol for using the HaloTag® system for ChIP in vivo, to map, with sensitivity and specificity, the cistrome of a dynamic mouse transcription factor expressed at its endogenous locus.

KW - ChIP

KW - Fusion protein

KW - HaloTag®

KW - NR1D1

KW - Nuclear receptor

KW - Tag

UR - http://dx.doi.org/10.21769/bioprotoc.4460

U2 - 10.21769/BioProtoc.4460

DO - 10.21769/BioProtoc.4460

M3 - Article

C2 - 35937930

SN - 2331-8325

VL - 12

JO - Bio-protocol

JF - Bio-protocol

IS - 13

M1 - e4460

ER -

HaloChIP-seq for Antibody-Independent Mapping of Mouse Transcription Factor Cistromes In Vivo

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Keywords

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Centre for Biological Timing

Rhythms in the beat: Circadian Clock Regulation of Cardiac Electrophysiology

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HaloChIP-seq for Antibody-Independent Mapping of Mouse Transcription Factor Cistromes In Vivo

Abstract

Keywords

Access to Document

Other files and links

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Centre for Biological Timing

Rhythms in the beat: Circadian Clock Regulation of Cardiac Electrophysiology

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