A synthetic biology approach to probing nucleosome symmetry
- University of Massachusetts Medical School, United States
- The Hebrew University, Israel
- European Molecular Biology Laboratory (EMBL), Germany
- Northwestern University, United States
Abstract
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read , we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read by effector proteins in the cell.
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Funding
National Institute of General Medical Sciences (R01GM100164)
- Yuichi Ichikawa
- Caitlin F Connelly
- Hsin-Jung Chou
- Hsuiyi V Chen
- Oliver J Rando
- Paul D Kaufman
European Commission (340712)
- Alon Appleboim
- Hadas Jacobi
- Nir Friedman
National Institute of General Medical Sciences (P41GM108569)
- Nebiyu A Abshiru
- Yupeng Zheng
- Paul M Thomas
- Neil L Kelleher
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Ichikawa et al.
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.
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A synthetic biology approach to probing nucleosome symmetry
eLife 6:e28836.
https://doi.org/10.7554/eLife.28836
