Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Summary

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Abstract

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Introduction

Mapping the binding sites of transcription factors (TFs), chromatin remodelers, and other chromatin-associated regulatory factors is key to understanding all chromatin-based processes. While chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) approaches have been used to gain many important insights into genome biology, they have notable limitations. We recently introduced an alternative method, termed chromatin endogenous cleavage and high-throughput sequencing (ChEC-seq)¹, to circumvent these drawbacks.

ChIP-seq is most often performed with an initial formaldehyde crosslinking step (X-ChIP-seq) ....

Protocol

1. Generation of Yeast Strains

1. Generate a yeast strain bearing the factor of interest tagged with MNase.
   1. PCR amplify the MNase tagging cassette from the desired vector (Table 1) using the specified reaction mixture (Table 2) and cycling conditions (Table 3). Mix 5 µL of the PCR reaction and 1 µL of 6X DNA loading dye. Run each PCR aliquot on a 0.8% agarose gel at 120 V for 40 min. The expected product size is ~2.3 kb.
   2. Transform the amplified tagging cassette into the strain of choice using standard lithium acetate transformation²² an....

Results

In the case of a successful ChEC experiment, analysis of DNA by agarose gel electrophoresis will reveal a calcium-dependent increase in DNA fragmentation over-time, as indicated by smearing and eventual complete digestion of genomic DNA. In some cases, a ladder of bands similar to that seen with a traditional MNase digestion is observed after extended digestion. This is the case for ChEC analysis of Reb1, a general regulatory factor that binds nucleosome-depleted regions (NDRs) (

Discussion

We have shown that ChEC can map diverse classes of yeast proteins on chromatin, and anticipate that it will be broadly applicable to different families of TFs and other chromatin-binding factors in yeast. ChEC-seq is advantageous in that it does not require crosslinking, chromatin solubilization, or antibodies. Thus, ChEC avoids artifacts potentially present in X-ChIP-seq, such as the hyper-ChIPable artifact^3,4, and native ChIP, such as false negatives due to inc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
dNTPs	NEB	N0447
Q5 high-fidelity DNA polymerase	NEB	M0491L	Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.
TrackIt 1 Kb Plus DNA ladder	ThermoFisher Scientific	10488085
Taq DNA polymerase	NEB	M0273L
cOmplete Mini EDTA-free protease inhibitor cocktail	Sigma-Aldrich	11836170001	It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit MNase cleavage by chelation of Ca2+.
PMSF	ACROS Organics	AC215740010
Digitonin, High Purity	EMD Millipore	300410-250MG	Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.
Proteinase K, 20 mg/mL	Invitrogen	25530049
RNase A, 10 mg/mL	ThermoFisher Scientific	EN0531
Ampure XP beads	Beckman Coulter	A63880	Ampure-like beads can be generated using a published protocol (ref 24).
MagneSphere magnetic rack	Promega	Z5342