PT  - JOURNAL ARTICLE
AU  - Yuichi Ichikawa
AU  - Caitlin F. Connelly
AU  - Alon Appleboim
AU  - Thomas C. Miller
AU  - Hadas Jacobi
AU  - Nebiyu A. Abshiru
AU  - Hsin-Jung Chou
AU  - Yuanyuan Chen
AU  - Upasna Sharma
AU  - Yupeng Zheng
AU  - Paul M. Thomas
AU  - Hsiuyi V. Chen
AU  - Vineeta Bajaj
AU  - Christoph W. Müller
AU  - Neil L. Kelleher
AU  - Nir Friedman
AU  - Daniel N. Bolon
AU  - Oliver J. Rando
AU  - Paul D. Kaufman
TI  - A synthetic biology approach to probing nucleosome symmetry
AID  - 10.1101/170811
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 170811
4099  - http://biorxiv.org/content/early/2017/09/06/170811.short
4100  - http://biorxiv.org/content/early/2017/09/06/170811.full
AB  - The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the 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 also 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 out by effector proteins in the cell.