A simple method for generating high-resolution maps of genome wide protein binding

  1. Peter J Skene
  2. Steven Henikoff  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States

Abstract

Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution, and newer techniques require significant experimental alterations and complex bioinformatics. Previously we have used a new crosslinking ChIP-seq protocol (X-ChIP-seq) to perform high-resolution mapping of RNA Polymerase II (Skene et al., 2014). Here we build upon this work and compare X-ChIP-seq to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity, to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base-pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing.

Article and author information

Author details

  1. Peter J Skene

    N/A, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Steven Henikoff

    N/A, Fred Hutchinson Cancer Research Center, Seattle, United States
    For correspondence
    steveh@fhcrc.org
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Joaquín M Espinosa, University of Colorado at Boulder, United States

Publication history

  1. Received: June 4, 2015
  2. Accepted: June 9, 2015
  3. Accepted Manuscript published: June 16, 2015 (version 1)
  4. Version of Record published: June 25, 2015 (version 2)

Copyright

© 2015, Skene & Henikoff

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 12,731
    Page views
  • 2,407
    Downloads
  • 65
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Peter J Skene
  2. Steven Henikoff
(2015)
A simple method for generating high-resolution maps of genome wide protein binding
eLife 4:e09225.
https://doi.org/10.7554/eLife.09225

Further reading

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics
    Claudia C Carcamo et al.
    Research Article Updated

    One-dimensional (1D) target search is a well-characterized phenomenon for many DNA-binding proteins but is poorly understood for chromatin remodelers. Herein, we characterize the 1D scanning properties of SWR1, a conserved yeast chromatin remodeler that performs histone exchange on +1 nucleosomes adjacent to a nucleosome-depleted region (NDR) at gene promoters. We demonstrate that SWR1 has a kinetic binding preference for DNA of NDR length as opposed to gene-body linker length DNA. Using single and dual color single-particle tracking on DNA stretched with optical tweezers, we directly observe SWR1 diffusion on DNA. We found that various factors impact SWR1 scanning, including ATP which promotes diffusion through nucleotide binding rather than ATP hydrolysis. A DNA-binding subunit, Swc2, plays an important role in the overall diffusive behavior of the complex, as the subunit in isolation retains similar, although faster, scanning properties as the whole remodeler. ATP-bound SWR1 slides until it encounters a protein roadblock, of which we tested dCas9 and nucleosomes. The median diffusion coefficient, 0.024 μm2/s, in the regime of helical sliding, would mediate rapid encounter of NDR-flanking nucleosomes at length scales found in cellular chromatin.

    1. Cancer Biology
    2. Chromosomes and Gene Expression
    Ariel Ogran et al.
    Research Article

    The transformation of normal to malignant cells is accompanied by substantial changes in gene expression programs through diverse mechanisms. Here, we examined the changes in the landscape of transcription start sites and alternative promoter (AP) usage and their impact on the translatome in TCL1-driven chronic lymphocytic leukemia (CLL). Our findings revealed a marked elevation of APs in CLL B cells from Eµ-Tcl1 transgenic mice, which are particularly enriched with intra-genic promoters that generate N-terminally truncated or modified proteins. Intra-genic promoter activation is mediated by (1) loss of function of ‘closed chromatin’ epigenetic regulators due to the generation of inactive N-terminally modified isoforms or reduced expression; (2) upregulation of transcription factors, including c-Myc, targeting the intra-genic promoters and their associated enhancers. Exogenous expression of Tcl1 in MEFs is sufficient to induce intra-genic promoters of epigenetic regulators and promote c-Myc expression. We further found a dramatic translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing metabolic stress. These findings uncovered the role of Tcl1 oncogenic function in altering promoter usage and mRNA translation in leukemogenesis.