Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Summary

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Abstract

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

Introduction

During the past decade, there has seen a dramatic improvement in sequencing technologies, allowing the study of a wide range of genomes in large numbers of samples, and with astonishing resolution. The Encyclopedia of DNA Elements (ENCODE) Consortium, a large-scale multi-institutional effort spearheaded by the National Human Genome Research Institute of the National Institutes of Health, has provided insights into how individual transcription factors and other regulatory proteins bind to and interact with the genome. The initial effort characterized specific DNA-protein interactions, as assessed by Chromatin immunoprecipitation (ChIP) for over 100 known DNA-binding pr....

Protocol

1. Capture Oligonucleotide Design

1. Design a panel of oligonucleotides to be used during the hybridization capture of the target region/s.
   1. Aim to design 4–8 oligonucleotides per target region, but as a minimum, design at least one oligonucleotide targeting each end of the target region.
   2. If the target sequence is long (>500 base pairs), design the oligonucleotides as spread out as possible to assure effective enrichment of chromatin across the entire target region.
      NOTE: We have observed optimal captures with 4–8 oligonucleotides. Even though this process can work well with more oligonucleotides,....

Results

Due to the need for large input amounts of chromatin for HyCCAPP to succeed, cells are grown to relatively high levels of confluency. Trypan blue staining is used to confirm that cell death rates are moderate (<10 %). In single copy experiments, chromatin content prior to hybridization capture needs to be in the femtomolar range, which usually requires at least 10⁹ cells as starting material. Prior to full scale experiments, it is recommended to test capture oligonucleotide .......

Discussion

The HyCCAPP method described here has many unique features that make it a powerful approach to uncover DNA-interactions that otherwise would remain elusive. The nature of the process gives HyCCAPP the flexibility to work in different organisms and regions of the genome. It is a method, however, that has several limitations to be considered.

HyCCAPP is a method that avoids any cell modifications so that it can potentially be applied in primary cells, cell culture systems, or even tissue samples.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PrimerQuest tool	IDT
OligoAnalyzer 3.1	IDT		Analyze capture oligonucleotids
PairFold	UBC		Analyze interactions
RPMI 1640 media	Thermo Fisher	11875093
Penicillin-streptomycin	Thermo Fisher	15140122
L-Glutamine	Thermo Fisher	25030081
Fetal bovine serum	Thermo Fisher	26140079
Countess automated cell counter	Thermo Fisher
850 cm2 roller bottle	Greiner Bio-one	680058
Roller system	Wheaton	22-288-525
37 % formaldehyde	Sigma-Aldrich	F8775
Glycine	Sigma-Aldrich
Igepal CA-630	Sigma-Aldrich	I3021
Protease inhibitor cocktail	Sigma-Aldrich	P4380
HEPES	Thermo Fisher	15630080
Branson digital sonifier SFX 150	Emerson
Qubit 3.0 fluorometer	Thermo Fisher
Qubit dsDNA BR assay kit	Thermo Fisher	Q32850
Bioanalyzer 2100	Agilent
Agilent DNA 1000 kit	Agilent	5067-1504
MES sodium salt	Sigma-Aldrich	M3885
NaCl 5M	Thermo Fisher	AM9760G
EDTA	Sigma-Aldrich
DynaMag-2 magnet	Thermo Fisher	12321D
DynaMag-15 magnet	Thermo Fisher	12301D
Dynabeads M-280 atreptavidin	Thermo Fisher	60210
Low binding tubes	Eppendorf	22431081
Hybridization oven	SciGene
Tube shaker and rotator	Thermo Fisher	415110Q
DNase I (RNase-free)	New England BioLabs	M0303
SSC buffer 20× concentrate	Sigma-Aldrich	S6639